#Taqman Probes

[eurofinsgenomics755]

Introduction to DNA Sequencing and TaqMan Probes

DNA sequencing is a fundamental technique used to determine the order of nucleotides in a DNA molecule, while TaqMan probes are specialized oligonucleotide probes employed in real-time polymerase chain reaction (PCR) assays for the detection and quantification of specific DNA sequences.

Understanding DNA Sequencing

Principles of DNA Sequencing

DNA sequencing relies on various methodologies to decipher the nucleotide sequence of a DNA molecule, providing valuable insights into genetic information and molecular structure.

Importance in Molecular Biology

DNA sequencing plays a crucial role in molecular biology research, enabling scientists to study genetic variation, identify mutations, and unravel the complexities of the genome.

TaqMan Probes: An Overview

Definition and Purpose

TaqMan probes are short, single-stranded DNA probes containing a fluorescent reporter dye and a quencher molecule. These probes hybridize to target DNA sequences during PCR amplification, generating a fluorescent signal proportional to the amount of amplified DNA.

Mechanism of Action

TaqMan probes work by binding to the target DNA sequence between the PCR primers. During PCR amplification, the Taq polymerase cleaves the probe, releasing the reporter dye and generating a fluorescent signal that is detected in real-time.

Applications of DNA Sequencing

Genome Analysis

DNA sequencing is widely used for genome analysis, enabling researchers to sequence entire genomes and study genetic variation, evolutionary relationships, and disease susceptibility.

Mutation Detection

DNA sequencing is invaluable for detecting mutations and genetic alterations associated with diseases such as cancer, inherited disorders, and infectious diseases.

Advantages of TaqMan Probes

Specificity and Sensitivity

TaqMan probes offer high specificity and sensitivity, allowing for the detection of target sequences with unparalleled accuracy and efficiency.

Real-Time PCR Applications

TaqMan probes are compatible with real-time PCR platforms, enabling the quantitative analysis of gene expression, SNP genotyping, and pathogen detection in real-time.

DNA Sequencing Techniques

Sanger Sequencing

Sanger sequencing, also known as chain termination sequencing, is a traditional DNA sequencing method that involves the synthesis of DNA fragments of varying lengths, followed by electrophoretic separation and detection of fluorescently labeled nucleotides.

Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) technologies enable high-throughput DNA sequencing, allowing for the rapid and cost-effective analysis of entire genomes, transcriptomes, and epigenomes.

Designing and Using TaqMan Probes

Probe Design Considerations

Designing TaqMan probes involves optimizing parameters such as probe length, GC content, and melting temperature to ensure efficient hybridization and specific target detection.

Experimental Applications

TaqMan probes find diverse applications in molecular biology research, including gene expression analysis, SNP genotyping, microbial detection, and viral quantification.

Challenges and Limitations

Cost and Throughput

High cost and limited throughput are significant challenges associated with DNA sequencing and TaqMan probe-based assays, particularly for large-scale genomic studies and diagnostic applications.

Probe Optimization

Optimizing TaqMan probe design and assay conditions is essential for maximizing sensitivity, specificity, and reproducibility, requiring careful experimental optimization and validation.

Conclusion

DNA sequencing and TaqMan probes have revolutionized molecular analysis, enabling researchers to explore the intricacies of the genome and detect specific DNA sequences with unprecedented accuracy and efficiency. By leveraging these technologies, scientists can advance our understanding of genetics and develop innovative solutions for diagnosing and treating genetic diseases.

[cytgen]

Abstract The hairpin probe is characterized by a higher fluorescence quenching efficiency as compared with the linear one under the conditions of real-time PCR, which leads to a lower background level of fluorescence and, consequently, a higher signal to noise ratio during real-time PCR. An experimental comparison of the fluorescence-quenching efficiency of two oligonucleotide probes in different conformations (hairpin in a molecular beacon format and linear in a TaqMan format) was made. There is a difference in the interaction between the quencher and fluorophore for the probes of different conformations. For a linear probe, quenching occurs through the mechanism of inductive-resonance energy transfer (Förster resonance energy transfer, FRET), while that for a hairpin probe quenching occurs through contact quenching through a closer arrangement of the fluorophore and quencher, but a resonant energy transfer according to the Förster mechanism is also possible. It was demonstrated that the absorption spectrum for the linear probe almost coincides with the absorption spectrum of an oligonucleotide representing a probe without a quencher, which indicates a dynamic (Förster) mechanism of energy transfer. On the contrary, the absorption spectra for the hairpin probe and oligonucleotide representing the probe without a quencher differ significantly, which indicates a contact mechanism of energy transfer between the fluorophore and fluorescence quencher. The fluorescence spectra of the probes and their complexes with the oligonucleotide complementary to the linear probe (and the loop of the hairpin probe) and the amplicon (200 bp in length containing a DNA target for the probes) allowed for comparison of these two probes by comparing the energy migration radii, the efficiency of donor fluorescence quenching. The energy migration radius R calculated by the experimental data was 32.4 Å for the hairpin probe and 47.3 Å for the linear probe.

[rnomics]

#cancers, Vol. 15, Pages 1271: Comparison of #RNA Marker Panels for Circulating Tumor Cells and Evaluation of Their Prognostic Relevance in Breast #cancer

Liquid biopsy is a promising tool for therapy monitoring of #cancer patients, but a need for further research in this field exists in order to improve sensitivity, specificity, standardization and minimize costs. In our present study, we evaluated two panels of transcripts related with the presence of circulating tumor cells (CTCs) (Panel 1: CK19, EpCAM, SCGB2A2 and Panel 2: EMP2, SLC6A8, HJURP, MAL2, PPIC and CCNE2) in two cohorts of breast #cancer patients (metastatic and early). A blood cell fraction possibly containing CTCs was isolated with density gradient centrifugation, followed by #RNA isolation and qPCR using TaqMan® or RT-qPCR using hybridization probes. The positivity rates of the investigated panels were similar, albeit higher in metastatic (69.4% Panel 1, 75.0% Panel 2; total 86.1%) compared to early (18.9% Panel 1, 23.3% Panel 2; total 31.1%) breast #cancer patients. CK19, SCGB2A2, EMP2, HJURP, MAL2, and CCNE2 individually correlated with shorter overall survival in the metastatic patient cohort. The findings highlight the additional value of Panel 2 markers, which are in contrast to CK19 and EpCAM not solely linked to an epithelial phenotype. https://www.mdpi.com/2072-6694/15/4/1271?utm_source=dlvr.it&utm_medium=tumblr

[cormiermilne]

Aggregation-Induced Emission Qualities within Totally π-Conjugated Polymers, Dendrimers, along with Oligomers.

M1 toxoid was tested as being a probable vaccine conjugate. Vaccination together with One to ten mu g M1 conjugated in order to ovalbumin (M1-ovalbumin) ended in faster along with quantitatively greater amounts of anti-ovalbumin IgG, together with endpoint titers becoming One particular,000- to be able to 12,000-fold more than those invoved with pets immunized with unconjugated ovalbumin. Substantially https://www.selleckchem.com/products/incb28060.html of anti-ovalbumin IgG have been seen in these animals transgenic regarding man main histocompatibility complicated (MHC) course Two. Alternative associated with M1 with the #link# MHC class II joining mutant (DM) eliminated superior health, suggesting that will M1 superior the supply of antigen through MHC class II-positive antigen-presenting tissue which most important inside lymphoid tissues. Immunization of pets which has a conjugate consisting of M1 and also ovalbumin peptide from opportunities 323 for you to 339 made amounts of anti-peptide IgG 100-fold higher than those involved with pets immunized together with peptide by yourself. Coupling of Proteasome inhibitor -defective superantigen toxoid gifts a whole new way of conjugate vaccines with all the added advantage of focused shipping and delivery to be able to MHC type II-bearing tissues #link# .An alternative records alternative involving SLC39A14, brought on by pre-mRNA splicing, had been recently proposed as being a biomarker pertaining to colorectal cancer malignancy (CRC). In your review, we now have authenticated the cancer-specific splicing routine in the mutually exclusive exons 4A and also 4B within altogether 244 colorectal tissue biological materials. Exon-specific quantitative RT-PCR examines across 136 Point IIV CRC examples and Forty-four normal colon mucosa trials demonstrated full cancer-specificity, and also 94% awareness involving SLC39A14-exon4B relative to SLC39A14-exon4A appearance. Even so, across Proto-oncogene tyrosine-protein kinase Src like 20 samples from your range of healthful cells, 20 #link# portrayed the actual CRC alternative. This became accurate also for 10 benign lymph nodes, indicating that the cancer-specificity is mainly restricted to the actual digestive tract along with anus. Consequently, clinical use of SLC39A14-exon4B as being a recognition marker pertaining to CRC other than in samples removed from the particular colon wall membrane weakens. Prognostic value by diagnosis associated with metastasis to be able to lymph nodes is also abated, elucidating an essential trap in order to biomarker breakthrough. However, looks at associated with ten nondysplastic biopsies coming from sufferers with energetic inflamation related colon ailment revealed unfavorable results in 7 examples and just weakly good results throughout 3 examples, advising price of SLC39A14-exon4B as a sign to distinguish CRC off their pathologic circumstances with the intestinal tract. To summarize, the actual SLC39A14-exon4B log variant is really a CRC biomarker with higher awareness and organ-confined nature. Even more standby time with the records and its particular protected health proteins isoform must be investigated within an organ-confined framework.A high throughput, real-time multiplex, solitary pipe RT-PCR assay was made for synchronised detection involving Spud leafroll malware (PLRV), Spud trojan By(PVX) and Potato malware Utes (PVS) in spud simply leaves along with tubers, and Tomato spotted wilt computer virus (TSWV) within potato tubers and tomato simply leaves. Quality utilizes a number of different fluorescently classed TaqMan (Ur) probes. Restrictions of detection awareness have been proven employing a range of trojan records backup quantities (8 x 12(One particular) to 8 x 15(Being unfaithful) replicates regarding PVX and PVS, My partner and i By 102 for you to My spouse and i times 1010 copies of PLRV along with One particular x 12(Three) to 1 by Ten(15) copies associated with TSWV). For everybody analysis, the particular inter-assay reproducibility had been substantial, having a coefficient involving variance of the combined assays regarding <2%. Total RNA had been removed quickly as well as successfully through bulked biological materials comparable to 300 dormant tubers to identify one attacks regarding PLRV, PVS as well as TSWV concurrently in a single analysis.

[tonkistealth]

Amplifx pcr

#AMPLIFX PCR SOFTWARE#

#AMPLIFX PCR CODE#

To validate this assay, serum samples were collected from patients infected, either acutely or chronically, with HBV or HCV and maintained at -20 Â☌ until use. Popular search Validation Of The Assay With Serum Samples All data is backed up multiple times a day and encrypted using SSL certificates.

#AMPLIFX PCR CODE#

Our internal code of conduct adds additional privacy protection. We use procedural, physical, and electronic security methods designed to prevent unauthorized people from getting access to this information. We’ve put industry-leading security standards in place to help protect against the loss, misuse, or alteration of the information under our control. You May Like: Hepatitis C And Liver Failure We Implement Proven Measures To Keep Your Data SafeĪt HealthMatters, we’re committed to maintaining the security and confidentiality of your personal information. The extracted DNA and RNA were stored at -80 Â☌ until use. The concentration and quality of the extracted DNA and RNA were assessed by Nanovue spectrophotometry and by amplification of a fragment of the gene coding for β-actin. Nucleic acids were extracted from 200 µL of serum using a QIAamp MinElute Virus Spin kit according to the manufacturer’s suggested protocol.

#AMPLIFX PCR SOFTWARE#

All of the primers were selected using the primer premier 5.0 software and were synthesized, purified and labeled by Takara Ltd. The fluorophore primers were quenched by partly complementary oligonucleotides of a single-base mismatched labeled with a quencher at 3′-end. The fluorophore primers and the reverse primers were used to amplify a 94-bp fragment in the highly conservative 5′ non-coding region of the HCV RNA. The baseline and threshold values were automatically adjusted for each test. Results were analyzed using 7500 Fast Software v. All reactions were performed in duplicate using universal conditions: 50 Â☌ for 2 min, 95 Â☌ for 10 min, 45 cycles of 95 Â☌ for 15 s, and 60 Â☌ for 1 min. The reactions were performed in a 7500 Real-Time PCR System using the TaqMan detection system with 12.5 µL of TaqMan Universal Master Buffer, predetermined concentrations of the primer-probe sets cited above, and 50 – 100ng of DNA or cDNA, for a total final volume of 25 µL per reaction. Blood Test : HCV RNA By Real Time PCR Quantitativeįor validation of the assay, the international panel cited above was used in qPCR reactions to construct standard curves of HBV DNA or HCV RNA in the following concentrations: HBV 7 or HCV 7, HCV 6 or HBV 6, HBV 5 or HCV 5, HBV 4 or HCV 4, HBV 3 or HCV 3, and HBV 2 or HCV 2.

[journalofsportsmedicine]

The Changes of Homocysteine Serum Level and Body Mass Index of Overweight Young Women after Eight Weeks of Pilates Exercise | Juniper Publishers

Juniper Publishers- Journal of Physical Fitness, Medicine & Treatment in Sports

Introduction

The increasing trend of obesity is one of the most major public health concern and health related issues [1]. According to an epidemiologic study conducted in 199 countries around the world in 2008, 1.46 billion adults were overweight. The scope of the outbreak obesity is varying substantially between nations, largest rise in Oceania and the lowest trend was estimated in India [1]. In the USA, 34 percent of adults aged above 20 suffered from obesity in 2008. in addition, East Mediterranean countries estimated an increased by over 20% [2]. A review study indicated that obesity reached an alarming level in all ages of the East Mediterranean countries. the spread development of obesity has been ranged from 25 to 81.9 percent among adults [3]. In the same vein, a research of systemic review has conducted a study regarding the effect of BMI on cardiovascular diseases which indicated that each 5 unit raised in BMI increases the chances of heart problems by 29 percent [4].

Homocysteine is considered to be associated with microalbuminuria, which is a serious indicator of the risk factor of future cardiovascular disease. Homocysteine is also known to mediate heart attack, whenever its level raises, the risk of arterial artery problems such as atherosclerosis would increase [5,6]. It is a phosphor containing amino acid which is formed during methionine metabolism [7]. Epidemiology studies revealed that high levels of homocysteine in blood plasma could be a risk factor for cardiovascular, heart attack and peripheral vascular diseases and it causes atherosclerosis through three ways of intra-arterial wall damage, interference in the blood-clotting factors, and the oxidation of low density lipoprotein [8]. The prevalence of hyper homo cysteinemia is estimated to be about 5 percent in the general population and 13 to 14 percent among patients with symptoms of atherosclerosis. However, these estimates are based on a cut above the 90th or 95th percentile of total homocysteine distribution in the general population [7,9]. Various surveys have shown that levels of this amino acid are at a high level in obese or overweight as well as those who do not have regular exercise. Yet, there is debate on the effects of physical activity on this factor [8].

The effect of stair climbing with moderate intensity exercise on cardio-respiratory Fitness, blood lipids, and serum homocysteine were examined among sedentary young women. This study showed that these exercises can favorably make some changes in homocysteine cardiovascular risk factors and blood lipids profiles of inactive young women [10]. Vincent also reported that the decrease in homocysteine level caused by resistive exercises among inactive elderly people in aged of 60_80 [11]. Moreover, no change in homocysteine level had been reported among 6 inactive men who participated in a walking activity with low intensity exercise [12]. In addition, another study showed that homocysteine concentration does not lead to significant changes in sub maximal exercise. Hence, the training intensity, sex and age are the factors affecting the above index [13]. Nevertheless, level of participation and different levels of interest in continuing exercise are the most important keys to reduce weight in overweight population although it may be exhausting. Therefore, doing sport activity such as Pilates can have a prominent role in increasing their interest in exercise (Table 1).  

A collection of specialized sport activities affecting body and mind by using special equipment is called Pilates. It can increase power or endurance of whole body and even target the deepest muscles. Pilates exercises include effort for one’s mental concentration on body muscles and how they function. Pilates exercise is named after its founder, Joseph Pilates, who developed a series of exercises in the 1920s to encourage physical and mental conditioning [14]. Strength, more body balanced, flexibility and core stability are emphasized in Pilates exercise to control of posture, movement, and breathing [15]. A survey in this domain showed that Pilates exercise among untrained women for 8 weeks leads to decreased serum creatine kinase, LDL, TG and Cholesterol. They also reported that Pilates increases HDL among these people [16]. Moreover, it has been investigated the effect of 24 weeks of Pilates exercise among elderly women and reported that this activity culminates in increased bone strength and decreased body lipid mass [17]. Nonetheless, there is no conclusive result about the effect of Pilates on homocysteine level and it has not been clearly identified whether the intensity employed in Pilates exercises has any effect on the amount of homocysteine of overweight women. Hence, the present research designed to investigate the effect of 8 weeks of Pilates exercise on protein and mRNA level of serum homocysteine and BMI among overweight young women (Table 2).

Participation and Method

Subject recruitment

 The study involved 20 overweight women whose BMI was above 25 from “Tandorosti gym” located in Tehran, Iran. The volunteers had the required features for participation in this project, including; no cardiovascular disease, not consuming special medicine, no smoking, not having a regular exercise. Physiologic characteristics of volunteers including height, weight, age, BMI, heart rate, systolic and diastolic blood pressure were recorded. Participations were well informed about the study prior to the experiment and written consent was obtained from them. A double-blind study method was applied, and the participants were randomly assigned into 2 groups of 10; (1) control and (2) Pilates exercise groups. Pilates exercise group performed Pilates exercise 3 times per week for 8 weeks. It contains movements which engaged abdominal muscles, hips, waist, legs and shoulder belt and it was performed on a mat without any specific equipment in three conditions of sitting, standing and lying down. During this period, the control group was also barred from participating in regular physical activity. Prior the exercise protocol and at the end of eighth weeks, BMI was recorded, and blood sample was collected to measure protein or mRNA level of homocysteine. All procedures involving experiments were carried out in strict accordance of the United States Institute of Research guidelines and approved by the Medical Centre Board of Tehran University (Table 3).

Method of body mass index measurement

 The body mass index was calculated by measuring height in meters, weight in kilograms and putting them in the following formula:

Height (m)2 / weight (in kilograms) = body mass index

Blood Sample Collection

Blood samples were collected at 24 hours before the pilates exercise and 24 hours after the last exercise session of 8 weeks training in fasting state through the elbow antecubital vein of all subjects for measurement of homocysteine mRNA and protein expressions.

Measurement of Serum Homocysteine protein

 The level of serum homocysteine protein was measured by homocysteine measurement Elisa kit (Axis-shield diagonistmade in Germany). ELISA was performed according to the manufacturer’s instructions. The absorbance for homocysteine was determined by using a microplate reader (iMark; Bio - Rad, Hercules, CA, USA) at a wavelength of 450 nm. A set of standard serial dilutions of known concentrations of homocysteine were provided by the manufacturer and were used to construct a standard curve in order to determine the homocysteine levels. Homocysteine which was attached to protein was changed to free homocysteine and then it was changed to S-adenosyl L homocysteine (Figure 1).

RNA purification and mRNA Expression Analysis by Real Time PCR (qPCR)

QIA amp RNA Blood Mini Kit (Qiagen, Germany) was used to isolate total cellular RNA from fresh whole blood. The concentration and purification of isolated RNA were evaluated by 260/280 UV absorption ratios (Gene Quant 1300, UK). Specific amplification fragments of DNA/RNA, Two-step Real time qPCR (quantitative Polymerase Chain Reaction) technique was used to calculate gene expression during the PCR amplification process with application of TaqMan reagent. This method was able to detect small differences between samples compared to other methods [18]. All reagents including probes and primers were obtained from Applied Biosystems, USA. TaqMan probe (known as fluorogenic 5′ nuclease) was chosen to perform qPCR. This probe has a sensitivity of 100% and a specificity of 96.67% [19] and is capable of detecting as few as 50 copies of RNA/ml and as low as 5-10 molecules [20].

Primers were designed by the same company for homocysteine targets

MRT: Hs01090026_m1; Lot no: 4351372, amplifies 61 bp segment from the whole mRNA length of 10558 bp. Beta Actin and GAPDH were used as reference genes. All amplification experiments were done in 3 biological replicates. Amplification program include 15 minutes at 48 °C (reverse transcriptase), 10 minutes at 95 °C activation of ampli Taq gold DNA polymerase, denaturation at 95 °C for 15 second and annealing at 60 °C for 1 minute. Denaturation and annealing steps were performed for 40 cycles. Step-One Plus real time PCR machine, TaqMan Fast Advanced Master Mix and assays were purchased from Applied Biosystems, USA. The fold changes of each target per average of ACTB were calculated and considered as mRNA expression levels of the target gene. Data was analyzed according to Comparative Ct (2−ΔΔCt) method, where amplification of the target and the reference genes were measured in the sample and reference [18].

    Statistical method

In this study, the Kolmogorov - Smirnov was used for the normal distribution of data as well as t-test (paired) for comparing pre-test and post-test stages within each group. Independent t-test was used for between-group comparison of indices in pre- and post-test stages. Pearson’s coefficient and linear regression were used to examine the association between indices. All data in significance level of 05.0 ≥P were examined using SPSS version 21 (Chicago, America) (Figure 2).

Results

BMI

 Findings of this research show that 8 weeks Pilates exercise leads to a significant decreased of BMI among overweight women (P=0.001).

Homocysteine protein expression quantification

The findings indicate that homocysteine protein expression levels were significantly increased after 8 weeks Pilates exercise among overweight women compared to control group or pretest (P=0.001).  

Homocysteine mRNA expression level

The results also indicate that homocysteine mRNA expression levels were significantly increased after 8 weeks Pilates exercise among overweight women compared to control group or pretest (P=0.0001). However, there was no significant change in the amount of these indices in the control group (P≤0.05). Also, there was no significant difference between the Pilates exercise group and the control group in pre-test stage regarding the homocysteine (P=0.004) and BMI (P=0.001) level.

    Discussion and Conclusion

This study reports a significant change in homocysteine level among overweight women after eight weeks of Pilates exercise. Although the comparison of two groups in the pre and post-test stages showed that the Pilates group had a lower level of homocysteine in the post-test stage compare to pre-test or control group, there was no significant change in the control group (P=0.004). A study investigated the effect of aerobic exercise on homocysteine level among overweight women which suffered from polycystic ovary syndrome showed that a 6-week aerobic exercise caused a significant decrease in the level of homocysteine [21]. In addition, similar study reported findings in young untrained women [10]. On the other hand, Di Santolo et al, investigated the relationship between recreational sports and homocysteine level among young women and found no significant change in the level of homocysteine [22].

The effect of two types of exercise on homocysteine level have been examined, as well as other cardiovascular diseases risk factors. It was revealed that a 12-week aerobic and resistive exercise does not make any significant change on the level of homocysteine, lipid profile and maximal aerobic power [23]. Furthermore, Bambaeichi et al, investigated the effect of increasing aerobic exercise on homocysteine level in young men and reported that the level of homocysteine did not have a significant change in the experimental group [24]. Various factors such as; intensity, type, or length of activity, gender or age of the participants have been considered to be effective on the divergent findings of the researchers. However, the effect of exercise on homocysteine can be explored from different aspects. During doing physical activity or recovery, amino acids more likely play an important role in anaplerotic functions sustaining the whole metabolic apparatus, as well as synthesis of other proteins, such as carnitine or melatonine. Increasing the energyreleasing reactions in the muscles; more induction in catabolism of amino acids such as methionine would be increasing. Also, regular sport activities increase renovation and repair muscle tissue by metabolic reactions require.

Since methionine is an amino utilized in protein formation which starting material for numerous biochemical molecules. On the other hand, homocysteine is one of the critical biochemical junctures between methionine metabolism and the biosynthesis of the amino acids’ cysteine and taurine, therefore decreasing methionine leads to homocysteine reduction [25]. There is this possibility that Pilates assists in decreasing homocysteine level and changing homocysteine to methionine leads to prevents its accumulation in the blood through increasing absorption of vitamins which are effective in homocysteine cycle especially group B vitamins (which decrease homocysteine during its menatbolosm) [26]. In addition, reduction of oxidative stress indices of regular exercise can also reduce levels of homocysteine [27]. Also, it is likely that insulin has an impact on homocysteine metabolism and its level through affecting activities of CBS, MTHFR enzymes, Systationine Gamma-lyase and betaine homocysteine methyltransferase [28]. Moreover, it seems that sport activities affect insulin role in decreasing homocysteine by reducing CRP and resistance to insulin [29].

The relationship between body composition and homocysteine is also of great importance. Although in the present study no significant relationship was found between them, BMI totally decreased with homocysteine reduction in Pilates group. The relationship between body composition and homocysteine have been investigated among Korean men and women in aged of 30 to 55 which had high homocysteine level. Findings of their study revealed that increase in total body fat content and decrease in LBM have a significant relationship with increased homocysteine. They reported that decrease in LBM is a suitable index for predicting homocysteine increase in different people [30]. Our findings showed that 8 weeks of Pilates exercise causes a significant decrease in BMI level among young overweight women. However, this change was not considered significant in the control group. In this domain, some consistent and contradictory studies can be mentioned. A 4 week of Pilates exercise on body composition have assessed in young girls. They showed that BMI level, waist size and blood pressure significantly decreased after 4 weeks of Pilates exercise [31]. Another study also explored the effect of two sport activities on indices of cardiovascular patients’ body composition. They declared that BMI level and waist-to-hip ratio significantly decreased compared to pre-test [23]. Alternatively, 5-week Pilates exercise on trunk power, endurance and flexibility among low active adult women had no any significant change in BMI level [32].  

 Furthermore, no significant change also caused after doing 8 weeks Pilates exercise on fitness indices among low-active women [33]. Similarly, Cakmakçi et al found no significant change of BMI as a result of 8 weeks of Pilates exercise among obese women [34]. It seems as if the amount of subcutaneous fat, exercise intensity and the amount of nutrition control of the participants are among the influential factors with contradictory findings. Different factors can be pointed out regarding the effective mechanisms on Pilates exercise. A negative relationship was reported between body composition and the level of VO2max [35]. It appears that sport activity can be effective in decreasing suitable BMI through increasing Lipoprotein Lipaz enzyme, fat tissue, blood flow, active muscles flow, and expanding the activity of hormones effective in metabolism [36-38]. In the similar vein, it has been reported a significant increase in aerobic power as a result of 8 weeks of Pilates exercise among obese women [39]. In conclusion, findings of this study showed that eight weeks of aerobic exercise significantly decreased the amount of homocysteine serum and body mass index in overweight young women. However, despite BMI level changes caused by Pilates, it seems that BMI decrease is not the major and effective factor on homocysteine decrease among overweight women. Probably there are other factors in this domain which have not been measured in this research. It is considered as the limitation of this study and should be taken into consideration in future researches.

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[ocelothouse89]

A Review On Effects Of Conjugated Linoleic Fatty Acid Cla Upon Body Composition And Energetic Metabolism

Responsibility for any adverse effects that may result from the use or application of the information contained on this website or on the Fast Metabolism Diet App is expressly disclaimed. It’s that conjugated linoleic acid that carries the alleged weight-loss benefits in CLA safflower oil supplements. So, CLA stands for conjugated linoleic acid – a fat high in monounsaturated and polyunsaturated fats (a.k.a. the good kinds). To examine Resveratone Diet , mice were subjected to a glucose tolerance test . Again, CR mice exhibited a marked improvement in glucose tolerance, an effect that could not be explained by increased insulin secretion, as plasma insulin levels at 30 minutes during the GTT were reduced . In contrast, glucose tolerance was not improved by 10,12 CLA, with no evidence of an insulin secretory defect . Unlike other products to help you lose weight, CLA does not have caffeine or similar ingredients. CLA works by increasing the rate at which you burn calories and improving your immune system. Ironically, it is found in many products that have a large amount of the unhealthy type of fat. RNA was extracted using an RNA extraction kit , reverse-transcribed, and cDNA amplified by qRT-PCR using an ABI 7900HT instrument. Taqman primer/probe sets for individual genes were from Thermo Fisher Scientific . Relative expression of target genes was calculated using the ΔΔCt formula and expressed as a fold change from HFHS-fed control EWAT. Over the years, researchers sought to examine whether similar effects could be found when applied to human subjects. For instance, research was conducted which included one control group and an experimental group (receiving 1.7 to 6.8 grams of CLA) for a period of 12 weeks. CLA is causing quite a stir because as of yet it does seem to be doing exactly what it claims it will do. That is help users shed excess pounds while preserving muscle mass. The occurrence of overweight and obesity has been augmented as the most common health issue of modern food style. Obesity is considered as a cause for many health problems such as heart diseases, infertility and insulin resistance . You might find it hard to believe, but it turns out that butter can be a fat-burning food! CLA has been shown to help with fat loss in many animal and some human studies, which is why in its concentrated form, it’s one of the most popular weight loss supplements in the world. A number of studies have found evidence that CLA not only reduces body weight and body fat mass, but may also help increase lean mass in different species. To gain insights into the mechanism whereby 10,12 CLA mediates its effects on energy expenditure to induce fat loss, we measured expression of key genes regulating lipid metabolism in WAT. The same goes for dairy products we get from cows, including cream or butter. One of the best ways to find high-quality grass-fed beef is to purchase it directly from small farms, whether visiting farmer’s markets, joining a community-sponsored agriculture group or even looking online. If you can’t find the perfect product, for example 100 percent organic and grass-fed beef, do the best you can while also focusing on limiting industrial and man-made fats from your diet. Consuming foods high in CLA or taking CLA supplements for 12 weeks seems to improve symptoms and overall well-being in people withseasonal allergy symptoms. Similarly, some research shows that for people with asthma, CLA might be a natural treatment method for asthma-related symptoms, due to its ability to help control inflammation. Twelve weeks of supplementation seems to improve airway sensitivity and ability to exercise. The browning of WAT, in which UCP1-positive cells are present within WAT depots, has recently been described as a potential mechanism for fat loss [reviewed by ], and can occur in response to physiological or pharmacological stimuli. With depot-specific alterations in FAO, a preferential metabolic pathway of brown adipocytes, we sought to determine if 10,12 CLA induces the browning of WAT. Ucp1 and Cidea gene expression was increased in both EWAT and IWAT , with higher UCP1 mRNA and protein detected in IWAT from 10,12 CLA-supplemented mice . However, expression of Fndc5, the gene that encodes irisin, was significantly elevated by 10,12 CLA in IWAT but not EWAT or BAT, providing a potential autocrine/paracrine mechanism of browning in this WAT depot. One of the first findings to bring conjugated linoleic acid into the health scene was its potential as a cancer prevention agent, especially in the fight against breast cancer. In animal studies, supplementing with CLA reduced the number of tumors in rats.

[healforceworld]

Real-time fluorescence quantitative PCR

The so-called real-time fluorescent quantitative PCR technology refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally quantitatively analyzing the unknown template through the standard curve.

Detection method

1. SYBRGreenⅠ method:

In the PCR reaction system, an excess of SYBR fluorescent dye is added. After the SYBR fluorescent dye is specifically incorporated into the DNA double-strand, it emits a fluorescent signal. The SYBR dye molecule that is not incorporated into the chain will not emit any fluorescent signal, thereby ensuring the fluorescent signal The increase in PCR products is completely synchronized with the increase in PCR products.

SYBR quantitative PCR amplification fluorescence curve

Melting curve graph of PCR product (single peak graph indicates the unity of PCR amplified product)

2. TaqMan probe method:

When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme cleaves and degrades the probe, making the reporter fluorescent group and quencher The fluorescent group is separated, so that the fluorescence monitoring system can receive the fluorescence signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed, and the accumulation of the fluorescence signal is completely synchronized with the formation of the PCR product.

[juniperpublishers-endocrinology]

Familial Nonmedullary Thyroid Carcinoma (Fnmtc): Molecular Advances with the New Sequencing Technologies- Juniper Publishers

Introduction

Approximately 3-9% of the thyroid neoplasms are hereditary. Familial non-medullary thyroid cancer (FNMTC) is diagnosed when three or more first-degree relatives are affected and is classified into Syndromic familial adenomatous polyposis, Gardner’s syndrome, Cowden’s disease, Carney’s complex type 1, Werner’s syndrome) and Nonsyndromic (thyroid cancer). Several candidate chromosomal loci and susceptibility genes have been reported but these results are not replicated in subsequent studies. As the Next Generation Sequencing (NGS) now offers a powerful new diagnostic approach of the goal of this article was to review the current knowledge the new approaches used for the molecular characterization of FNMTC cases.

Familial predisposition of nonmedullary thyroid carcinoma (FNMTC) is diagnosed when three or more first-degree relatives are affected. It occurs in about 3-9% of the follicular cell-derived neoplasms and is classified into Syndromic and Nonsyndromic because encompasses a heterogeneous group of diseases [1]. The Syndromic group is characterized by a predominance of non-thyroidal tumors. Some syndromes are more frequently observed, such as familial adenomatous polyposis (FAP), Cowden syndrome, Werner syndrome and Carney complex, with thyroid cancer prevalence of 2-12%, 35%, 18% and 15%, respectively. Except for Werner’s syndrome, all syndromes related with FNMTC are autosomal dominant. Several driver-genes have been identified, APC gene was associated to FAP and PTEN, SDH, PIK3CA, AKT1 and KLLN were related to Cowden syndrome; PRKAR1α was related to Carney complex and WRN gene was associated to Werner’s syndrome [1].

The Nonsyndromic form accounts for 95% of all FNMTC cases and is characterized by the presence of thyroid cancer and the absence of other known associated syndromes [2]. The Papillary Thyroid Carcinoma (PTC) subtype is the most commonly observed and may or may not be associated with benign thyroid neoplasms (multinodular goiter) or autoimmunity thyroid disease (Hashimoto’s thyroiditis). Several candidate chromosomal loci and susceptibility genes have been reported (1q21, 2q21, 6q22, 8p23.1-p22 and 19p13.2, NKX2‐1, FOXE1, SRGAP1, TERT, HABP2 and C14orf93) suggesting that it is a polygenic familial cancer syndrome [1,3-5]. As subsequent studies failed to identify segregation of these candidates with the disease and due to the clinical variability of the patients, the molecular profile of each FNMTC family may be unique [6-9]. Furthermore, important cancer-related genes (APC, PTEN, TSHR, RET, TRK, c‐MET, BRAF and H‐K‐N‐RAS) were also excluded as the cause of FNMTC [10]. In the last decade, techniques for single-target detection have been replaced by the Next Generation Sequencing (NGS), which allows simultaneous analysis of a large group of genes generating a large volume of data in parallel [11-13]. The NGS high-throughput platforms are more efficient, less expensive and provides information that is not provided by Sanger DNA sequencing analysis or by hot spot mutation gene targeted assays (MLPA - Multiplex Ligation Probe-dependent Amplification or Taqman Genotyping) [10–12]. Analysis of the generated data is a complex process. In familial diseases with clinical heterogeneity, as observed in FNMCT, a careful selection of the individuals to be submitted to the NGS is necessary. The inclusion of unaffected individuals improves diagnostic rates by facilitating variants selection and mutations identification due to the exclusion of regional polymorphisms. The definition of a suitable pipeline for the identification and classification of the variants is also an essential step [11,12]. In silico predictive analysis provided by specific programs such as Polyphen2, SIFT, Mutation Assessor and Mutation Taster is a valuable tool to distinguish between the pathological and benign variants associated with the phenotype, taking into account the evolutionary conservation of the amino acid or nucleotide residue, biochemical impact of the amino acid substitution considering the physicochemical properties, and the variant localization [11,12]. As there is no rule, the use a more stringent filtering criterion are more appropriate as the first approach while less strict selection may be adequate when no candidate variant was identified [13-16]. It is noteworthy that a consensus among the prediction results leads to a better accuracy, as in CONDEL, PON-P and Meta-SNP algorithms [11,17,18]. Information obtained from population databases (1000 Genomes Project, ExAc and Exome Variant Server (EVS)) are frequently used to exclude variants that are deemed polymorphic/benign based on a global minor allele frequency (MAF) cut off of ~1% (0.01).

However, it is important to consider ethnicity-specific MAFs that depend on the ethnic background of the population, particularly in populations with high miscegenation as observed in Brazilians, underrepresented in most of the genomic databases, however data made available by local consortiums as ABraOM [19] and EPIGEN-BRAZIL [20] unravel this problem. In the literature, it is already possible to observe the benefits of new generation sequencing in the FNMTC. Recently, a group sought to identify previously undescribed cancer-predisposing gene(s) in a Cowden Syndrome family enriched for thyroid cancer across 4 generations, who had tested negative for PTEN, SDHB-D and KLLN, via an approach combining exome sequencing and family study. They found a variant in SEC23B gene and suggested that this germline heterozygous variant is associate with cancer predisposition [21]. Moreover, when whole-genome approach was conducted in sporadic FAP patient in which any pathogenic APC mutations was found by the conventional Sanger sequencing, a mosaic mutation in ~12% of his peripheral leukocytes was identified. Demonstrating that NGS is an effective tool to identify genetic mosaicism in hereditary diseases [22]. A variant on WRN gene was identified by exome-wide sequencing in a 16-year-old girl with an atypical syndrome without diagnosis after 10 years of the first symptoms. The variant has been previously reported in Werner’s syndrome (WS) and when the patient was re-evaluated several features of WS were detected, allowing the early diagnosis of a recessive disease and the early use of adequate therapies and interventions [23]. Using Whole Exome Sequencing (WES) a germline variant in the HABP2 gene was identified in a family with seven PTC patients of a FNMTC kindred and in 4.7% of 423 sporadic PTC cases. This variant increased protein expression in the tumor samples and functional studies showed that the loss of function p.G534E rs7080536 variant leads to increased colony formation and cell migration, characteristics of malignant transformation, suggesting that the HABP2 gene may be involved in susceptibility to thyroid cancer [4]. Because the authors did not report the ethnicity of the family and for having used only one database to determine MAF (1000 Genomes), this result was questioned by others groups [24]. Furthermore, this variant was considered a polymorphism in other populations (United Kingdom, USA, Saudi Arabia, Colombia, Spain, Italy, Australia and Brazil) [24].

Only Zhang et al identified the rs7080536 variant in 4/29 (13.8%) of unrelated FNMTC kindreds [25]. In other study, through linkage analysis and exome-sequencing the genes C14orf93 (RTFC), PYGL and BMP4 were identified as candidate genes in a FNMTC family with 5 cases of PTC. However, the functional studies showed that only the p.V205M mutation of the C14orf93 gene led to increased migration rate and colony formation in a PTC cell line [5]. Next Generation Sequencing allied to capture of expressed sequences from genomic DNA now offers a powerful new diagnostic approach. Barriers to use this technology still include cost and the complexity of interpreting results arising from simultaneous identification of large numbers of variants. Thus, cost reductions and new friendly analysis of this big data are necessary. Even so, the results obtained through this new generation sequencing technology have opened doors to new possibilities in the search for the molecular bases of thyroid familial cancer.

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[evoldir]

Fwd: Graduate position: JagiellonianU.EvolutionaryBiology

Begin forwarded message: > From: evoldir@evol.biology.mcmaster.ca > Subject: Graduate position: JagiellonianU.EvolutionaryBiology > Date: 11 July 2020 at 07:10:08 BST > To: rdmpage@gmail.com > Reply-To: brian@helix.biology.mcmaster.ca > > > Graduate position:Krakow_JagiellonianU.Evolutionary_Biology > > > PhD position in Evolutonary Biology > PhD student position is offered from October 2020 within the Polish > National Science Centre grant Environment-dependent balancing selection in > a gene involved in sexual conflict in Genomics and Experimental Evolution > Group at Jagiellonian University, Institute of Environmental Sciences. > > The project > The maintenance of genetic variability, enabling populations to adapt > to novel environments, is one of the greatest puzzles in evolutionary > biology. This is because ubiquitous directional selection should lead to > depletion of genetic variation in selected traits. This is especially the > case with sexually selected traits, in which directional selection is > particularly strong. Yet, substantial genetic variance in these traits > is maintained. A potent force proposed to maintain genetic variation is > balancing selection which can take a form of a crossover genotype by > environment interaction for fitness in heterogeneous environments. It > causes selection to act in environment-dependent manner so that one > allele is favored in one environment and the other at another one. > We aim to investigate the maintenance of polymorphism in  Phosphogluconate > dehydrogenase (6Pgdh) ?a sexually selected gene associated with sexual > conflict in the bulb mite Rhizoglyphus robini. 6Pgdh polymorphism (with > two alleles, S and F) is associated with differences in male reproductive > success. The S-bearers have advantage in male-male competition, but > decrease fecundity of their partners. Previous studies suggest that 6Pgdh > polymorphism is maintained by environment-dependent balancing selection, > but the exact mechanisms driving this selection are unknown. PhD candidate > will investigate ecological factors that determine persistence of the > polymorphism. > > Scope of work > PhD candidate will assess the level of 6Pgdh polymorphism in natural > populations and determine environmental factors affecting 6Pgdh allele > frequencies in the field. He/she will conduct experimental evolution > and will be involved in phenotypic measurements in the lab that will > enable direct test of the role of potential factors driving 6Pgdh > frequencies. Real-time PCR with TaqMan probes will be used to genotype > individuals. PhD candidate may also be involved in other molecular > analyses conducted in frames of the project, including transcriptomics. > > > Place and salary > The Student will join a dynamic, cooperative research group at the > Institute of Environmental Sciences, Jagiellonian University, Krakow, > Poland (www.eko.uj.edu.pl/en_GB).  The Institute of Environmental Sciences > is one of the most influential and best-recognized research institutions > in the fields of Ecology and Evolution in Central Europe located in > a beautiful medieval city with rich history and lively cultural life, > well connected to other European cities. > > The PhD student  will receive a tax-free scholarship from doctoral school > (ca. 2500 PLN) and/or a tax-free research stipend from the National > Science Centre grant (3000 PLN per month). > > Requirements > > The successful candidate will have a M.Sc. degree in biology or other > relevant fields by the start of the studentship. We are looking for a > student with good English, strong background in Evolutionary Biology > and experience in molecular techniques as well as good skills in data > analyses. Excellent communication and organizational skills are also > required. > > Documents > > Please send a CV including contact details for two references and a > cover letter to Agata Plesnar-Bielak (agata.plesnar@uj.edu.pl) by August > 10. The selected candidate will be assisted with a formal application > to the PhD program at Jagiellonian University (the exam will take place > between 9th and 14th September 2020) > > For more information, please e-mail Agata Plesnar-Bielak. > > > Agata Plesnar-Bielak > via IFTTT

[biotechtimes]

A Blueprint To Develop A Rapid qRT-PCR kit To Detect SARS-CoV-2: Study

New Post has been published on https://biotechtimes.org/2020/07/14/a-blueprint-to-develop-a-rapid-qrt-pcr-kit-to-detect-sars-cov-2-study/

A Blueprint To Develop A Rapid qRT-PCR kit To Detect SARS-CoV-2: Study

ACTREC Mumbai based scientists have presented a rapid, accurate and easy to implement qRT-PCR kit based assay to Detect SARS-CoV-2

By Ratneshwar Thakur

New Delhi, July 13, 2020 (SciSoup): Amidst the COVID-19 pandemic, within a span of about 3 months, over and above 8.78 lakhs positives cases and more than 23K death have been reported in India. Thus, radical measures are needed to flatten the rate of growing infection. The development of diagnostic kits to perform rapid, accurate test with low false-negative RT-PCR results in a cost-effective manner — may help in the early implementation of epidemiological containment measures.

Now a research team led by Dr. Amit Dutt at Advanced Centre for Treatment, Research, and Education in Cancer (ACTREC), Navi Mumbai – have published a report in the Cell Press journal ‘Heliyon’ where they present a rapid, accurate and easy to implement real-time PCR based assay with automated analysis at a cost of under $3 per reaction and turnaround time of less than 2hour. The study may facilitate faster in-house testing across laboratories to detect SARS-CoV-2.

“We have identified a unique conserved region in the SARS-CoV-2 Nucleocapsid gene. Using molecular biology techniques, we have generated synthetic DNA and RNA molecules for 3 such regions and spiked in the human genomic DNA and total RNA at varying concentrations to generate synthetic reference sample mimicking a true positive COVID-19 sample for the study. The standardizations performed were validated using COVID 19 positive and negative clinical specimens,” explained Dr. Amit Dutt, a scientist at ACTREC, Navi Mumbai, and corresponding author in this study.

In this study, the reference sample was used to compare the detection limit of two different chemistries (molecular biology methods): a probe-free SYBR Green dye-based methodology and a TaqMan probe-based RT-PCR assay. The probe-based assay was found to be more sensitive with detection of up to 15 RNA copies, as compared to 150 RNA copies of the probe-free method.

This was further developed into a one-step, one-tube, multiplex diagnostic kit. The detection accuracy of the kit was evaluated using a panel of 26 clinical specimens by performing 184 validations with dilutions of samples ranging from 1-100 ng.

Bhasker Dharavath and Neelima Yadav, ACTREC based researchers and co-first author of the study, say the kit was found to be 100% specific and 100% sensitive under limited settings based on accurate identification of 5 positive samples out of 26 samples and calling rest 21 negative samples as negative. The actual accuracy can only be established with a larger sample set, laments Bhasker and Neelima.

Furthermore, to minimize variability and automate the qRT-PCR downstream analysis of multiple samples in the same run, they developed an open access freely available GUI based analytical tool COVID qPCR Analyzer tool (www.actrec.gov.in/pi-webpages/AmitDutt/Covid/Analyzer.html).

“This is a novel automated computational tool to detect the virus in an unambiguous and reproducible manner with minimal requirement of third-party tools, and applicable to all kinds of real-time PCR machine for COVID detection,” says Sanket Desai, another co-first author of the study.

“Our study would empower researchers across research institutes to build their own capacity for developing a highly accurate and validated diagnostic kit to detect SARS-CoV-2 infection at a fraction of cost, in an Atmanirbhar way,” said Dr. Dutt.

The research team included Bhasker Dharavath, Neelima Yadav, Sanket Desai, Roma Sunder, Rohit Mishra, Madhura Ketkar, Prasanna Bhanshe, Anurodh Gupta, Archana Kumari Redhu, Nikhil Patkar, Shilpee Dutt, and Sudeep Gupta and Amit Dutt. (SciSoup – A Science and technology blog)

Journal Reference:

A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2

[juniperpublishers-rr]

MicroRNA-New Category of Diagnostic Biomarkers- Juniper Publishers

Introduction

MicroRNAs (miRNA) are small (21-24 nucleotides) non-coding RNAs which are involved in negative posttranscriptional regulation of gene expression. One miRNA molecule can have up to hundreds of target mRNAs and one mRNA can be regulated by multiple miRNAs, creating a complex regulatory network in the body/organism [1].Changes in intracellular miRNA levels affect gene expression, alter cell signaling, and significantly affect cellular metabolism. They regulate physiological processes including cell differentiation, regeneration and apoptosis, as well as pathological processes such as neoplastic transformation of cells. Therefore, it is not surprising that aberrant miRNA expression is involved in the pathogenesis of many diseases including cardiovascular diseases, neurodegenerative diseases and cancer. To this day, more than 2,500 different microRNAs have been described in various documents [2-7].

In addition to the intracellular occurrence, microRNAs were also detected in the extracellular area - in the blood and its derivatives (plasma, serum), but also in the urine, saliva and other body fluids. 5,6 It has been proven that levels of circulating microRNAs are not random and reflect the events occurring within the organism/body. The gathered data, together with the proven high miRNA stability, place the circulating miRNA into the role of potentially useful, clinically relevant biomarkers. Finding specific miRNAs or groups (profiles) of several miRNAs at once, in order to serve as diagnostic or prognostic sets, is now one of the main targets of research in this area [2-8].

Methods

Detection and quantification of small non-coding RNAs is a challenging and interesting topic of contemporary science. Not only a small length of these molecules (about 25 nucleotides) and their low concentration in body fluids, but also the existence of sequence differences in unit bases in some types of short RNA (isomiRs), are key points for using these molecules as markers in routine diagnostics. The most common techniques for detecting small RNAs can be divided into many groups, and their combinations: Polymerase Chain Reaction (PCR), Microarrays, New Generation Sequencing (NGS), etc. [9-11]. Recently, a new immunomethod for detection miRNA was described.

Results

Detection technology based on PCR

Since the discovery of reverse transcription PCR (RT PCR), PCR-based systems are the most widely used methods for detecting RNA sequences. In this technology, the RNA molecule is first enzymatically transcribed (via the reverse transcriptase enzyme and the RT primer) into the cDNA sequence, which is then amplified by DNA polymerase using two specific primers. The PCR reaction can be visualized in real time (qRT PCR) using a sequence non-specific (Sybergreen) or sequence-specific (TaqMan system) fluorescent labels/markers. Unfortunately, in the context of short RNA detection, this standard qRT PCR system is not well suited because the RT primers do not have enough space to bind the target RNA due to their short length. Therefore, new approaches to the detection of short RNA molecules have to be applied, such as using a special RT primer, amplification of circular molecules (RCA), poly-tailing reaction or ligation reaction. So far, several methods of quantification of miRNA based on qRT PCR have been developed, and many of them are also commercially available[12, 13].

Detection technology based on microarrays

Microarrays (DNA / RNA chips) use a hybridization process that naturally parallels complementary DNA and RNA sequences. In case of a microarray, a synthetic DNA probe is bound to the defined position of a solid surface, designed to complement the sequence observed. Many DNA probes can be bound on a small surface, and different colors of fluorescent labels can be used to visualize hybrids. This reveals the main strength of micro multiplexing. The latest commercial detectors of miRNA (GeneChip miRNA 4.0 Arrays - ThermoFisher Scientific, miRCURY LNA microRNA arrays - EXICON)[14] are capable of measuring hundreds of miRNAs in one sample, plus miRNA targets can be selected according to customer needs. In principle, microarrays should provide highly quantitative information on nucleic acid concentration. In practice, however, there are significant quantitative bias between different genes and transcripts. As a result, microarrays are typically considered as a tool to relative quantification of different miRNA targets rather than to determine absolute concentration. Additionally, sensitivity of detection of microarrays is limited by sensitivity of the fluorescence detector, which means that very expensive devices are needed. For these reasons, microstructures are mainly used for differential analysis of profiles of miRNA expression profiles, but not for normal/routine measurements of concentration [15- 16]own experiences.

NGS

NGS is a group of approaches for massively multiplexed sequence analysis of DNA and RNA. Unlike traditional Sanger sequencing, which requires an input in form of a homogenous DNA template, NGS allows the analysis of heterogeneous samples while providing sequential information for more than 10 million randomly selected molecules of nucleic acid within the sample. Because of the large quantity of reads, NGS is uniquely suited for the analysis and diagnosis of nucleic acids requiring multiplex analysis of many genes and their variants. There are several NGS accesses and compatible devices on the market. The most common is Illumina, which is based on the sequential addition of fluorophore marked nucleotides in combination with fluorescence imaging. NGS is the most sensitive method of miRNA identification and quantification. The advantage of this technology is the possibility to discover new short RNAs that have not yet been described. However, the whole NGS process is very time-consuming and requires a high volume of samples. It is also very expensive - it requires chemicals, instruments/ equipment and highly sophisticated evaluation software[17-18].

Immuno-method for the detection and quantification of microRNA

A substantial number of molecules, including microRNAs are reported as disease markers for various human pathologies. Only a small fraction will be translated to clinical routine use. Major challenge is the reliable measurement of novel markers on platforms that are commonly used in clinical laboratories. Although current molecular methods used to measure miRNA biomarkers, such as qRT-PCR and NGS, are available in selected clinical laboratories, they are rather expensive. Moreover, compatibility with clinical high-throughput workflows is challenging. The adaption of miRNA assays to platforms and technologies that would overcome those issues may foster their use. One of the technologies, which overcome the problems of the current test formats mentioned above, is immunoassay platform, which has a short time-to-result and is faster than NGS. At the same time, the assay is inexpensive. Among the most important points with respect to immunoassay is that the required hardware is installed in many central laboratories of hospitals worldwide.

Immunoassay platforms are already routinely used in clinical laboratories worldwide, and many immunological tests are carried out on these commercial systems. Immunoassays are also adaptable to immunoassay analyzers. The general principle of immunoassay technique is detection or quantification of analyte (microRNA) in the sample comprising an immune reaction between an antibody and an analyte [19].

Discussion

Immunoassay platforms are already routinely used in clinical laboratories worldwide, and many immunological tests are carried out on these commercial systems. Immunoassays are also adaptable to immunoassay analyzers. The general principle of immunoassay technique is detection or quantification of analyte (microRNA) in the sample comprising an immune reaction between an antibody and an analyte.

In conclusion, we presented new innovative technique for quantification of miRMA based on ELISA. These methods are cheap and can be use in routine laboratories.

For more Open Access Journals in Juniper Publishers please click on: https://juniperpublishers.com 

For more articles in Open Access Journal of Reviews & Research please click on: https://juniperpublishers.com/arr/index.php 

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[missmiumiu]

Anal Chem. 2018 Jan 16;90(2):1273-1279. doi: 10.1021/acs.analchem.7b04050. Epub 2018 Jan 3.

Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.

Kim SC1, Clark IC1, Shahi P1, Abate AR1,2,3.

Author information

1Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco , San Francisco, California, United States.

2California Institute for Quantitative Biosciences (QB3), University of California-Berkeley , Berkeley, California, United States.

3Chan Zuckerberg Biohub , 499 Illinois Street, San Francisco, California, United States.

Abstract

Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.

＋

Cells & chemicals

Jurkat and MCF7 cells are cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) and antibiotics at 37°C in the presence of 5% CO2. 0.8–1 million Jurkat/MCF7 cells are collected and washed with cold PBS supplemented with 2% FBS (PBS-F). Cells are centrifuged at 300 RCF for 3 minutes, resuspended in 100 μL of 10 μM calcein violet (Thermo Fisher, C34858) or 25 μM calcein red (C34851) in PBS-F, and incubated on ice for 30 minutes. After cell staining, cells are pelleted, resuspended in 100 μL of PBS-F, mixed together and supplemented with 40 μL (0.2× volume) of Optiprep (Sigma, D1556) for density matching. The alkaline lysis buffer (ALB) contains 200 mM NaOH, 60%(v/v) PEG-200 and 2%(v/v) Triton X-100. It is noteworthy that the addition of PEG-200 increases pH significantly,25thus enabling the use of a lower NaOH concentration during the lysis step. This is important so that the buffer capacity of RT-PCR reagents is sufficient to effectively neutralize the alkaline after the merge step. Water-in-oil droplets are generated in a fluorinated oil (3M, HFE Novec 7500) supplemented with 2%(w/w) 008-FluoroSurfactant (RAN Biotechnologies). The oil phase of collected droplets is swapped with the FC-40 oil (Sigma, F9755) supplemented with 5%(w/w) 008-FluoroSurfactant immediately before thermocycling droplets for RT-PCR. The RT-PCR mix is composed of 1× Reaction Mix and 1× Enzyme Mix (Thermo Fisher, 12574030, SuperScript III One-Step) supplemented with 2.5%(v/v) Tween 20 and 2.5%(v/v) PEG 6,000. The FAM-labeled TaqMan probe specific to CD45 protein (IDT PrimeTime, Assay ID: Hs.PT.53a.2558434) is added to the RT-PCR mix.

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[rnomics]

Toxins, Vol. 10, Pages 431: Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats

Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10−8 ng μL−1. The anaC assay and a cyanobacterial specific 16S #rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R2 = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to #RNA assist in understanding anatoxin production. http://bit.ly/2PqticR

[awesomechrisharry]

Real Time PCR Fluorescent Probe Market 2021 In-depth Analysis, Growth with Forecast 2027: ThermoFisher, BioCat, Sigma-Aldrich, Biolegio, Glen Research

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