Circulating cell-free microRNAs are promising candidates for minimally invasive clinical biomarkers for the diagnosis, prognosis and monitoring of many human diseases. Despite substantial efforts invested in the field, the research so far has failed to deliver expected results. One of the contributing factors is general lack of agreement between various studies, partly due to the considerable technical challenges accompanying the workflow. Pre-analytical variables including sample collection, RNA isolation, and quantification are sources of bias that may hamper biological interpretation of the results. Here, we present a Two-tailed RT-qPCR panel for quality control, monitoring of technical performance, and optimization of microRNA profiling experiments from biofluid samples. The Two-tailed QC (quality control) panel is based on two sets of synthetic spike-in molecules and three endogenous microRNAs that are quantified with the highly specific Two-tailed RT-qPCR technology. The QC panel is a cost-effective way to assess quality of isolated microRNA, degree of inhibition, and erythrocyte contamination to ensure technical soundness of the obtained results. We provide assay sequences, detailed experimental protocol and guide to data interpretation. The application of the QC panel is demonstrated on the optimization of RNA isolation from biofluids with the miRNeasy Serum/Plasma Advanced Kit (Qiagen).
- MeSH
- Cost-Benefit Analysis MeSH
- Biomarkers blood MeSH
- Circulating MicroRNA blood isolation & purification MeSH
- Rats MeSH
- Real-Time Polymerase Chain Reaction economics instrumentation methods standards MeSH
- Humans MeSH
- Reagent Kits, Diagnostic standards MeSH
- Quality Control * MeSH
- Feasibility Studies MeSH
- Healthy Volunteers MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Glutathione S-transferase M1 is a cytosolic enzyme important for the biotransformation of xenobiotics in various human tissues. The aim of this study was to perform genetic analysis of deletion polymorphism in the GSTM1 gene by using the technology of digital PCR. For genotyping, the QX100 Droplet Digital PCR System was used. The absolute quantity of GSTM1 copies was normalized to the ß-globin reference gene. In our experimental group, the prevalence of GSTM1*0 null variant was 67 %. Frequencies of GSTM1*1/*1 (n=5), GSTM1*0/*1 (n=23), and GSTM1*0/*0 (n=22) genotypes were 10 %, 46 %, and 44 %, respectively. Digital PCR seems to be an available and reliable technology for GSTM1 deletion polymorphism genotyping.
- Keywords
- digitální PCR,
- MeSH
- Gene Deletion MeSH
- Molecular Diagnostic Techniques methods MeSH
- Adult MeSH
- Genetic Predisposition to Disease * MeSH
- Genotyping Techniques * methods instrumentation MeSH
- Clinical Studies as Topic MeSH
- Real-Time Polymerase Chain Reaction methods instrumentation MeSH
- Middle Aged MeSH
- Humans MeSH
- Neoplasms diagnosis genetics MeSH
- Computers utilization MeSH
- Polymerase Chain Reaction * methods instrumentation MeSH
- Polymorphism, Genetic MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.
- MeSH
- DNA, Neoplasm genetics MeSH
- DNA Topoisomerases, Type I genetics MeSH
- Nitrogen MeSH
- Tissue Fixation methods MeSH
- Carcinoma chemistry surgery MeSH
- Colon chemistry MeSH
- Cryopreservation instrumentation methods MeSH
- Real-Time Polymerase Chain Reaction instrumentation methods MeSH
- Humans MeSH
- Neoplasm Proteins biosynthesis genetics MeSH
- Colonic Neoplasms chemistry surgery MeSH
- Preservation, Biological instrumentation methods MeSH
- Specimen Handling instrumentation methods MeSH
- Gene Expression Regulation, Neoplastic MeSH
- Reproducibility of Results MeSH
- DNA, Ribosomal genetics MeSH
- Quality Control MeSH
- RNA, Neoplasm analysis genetics isolation & purification MeSH
- RNA, Ribosomal, 18S genetics MeSH
- Organ Preservation Solutions MeSH
- High-Throughput Screening Assays instrumentation methods MeSH
- Gene Expression Profiling methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Here we report one of the smallest real-time polymerase chain reaction (PCR) systems to date with an approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in the form of virtual reaction chambers (VRCs) where a ≈200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate the PCR performance. The standard curve slope was -3.02 ± 0.16 cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of 0.91 ± 0.05 per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics.
Undercooked lamb and mutton are common sources of Toxoplasma gondii infection for humans. A sequence specific magnetic capture technique in combination with quantitative real-time PCR targeting the 529 bp repeat element of T. gondii was used for estimation of the parasite burdens in various sheep tissues (n = 6) three months after peroral experimental inoculation with 10,000 T. gondii oocysts. Brain was the most frequently affected organ (positive in all 6 sheep) and showed the highest estimated parasite loads (0.5-30,913 parasites/g tissue). Lung samples were positive in three sheep, with load estimates of 36.3 to <1 parasite/g tissue. Heart tissue was positive in three sheep and kidney only in one animal with low parasite loads (<1 parasite/g tissue). Only few skeletal muscle samples in 2 animals showed positive results, with very low parasite burdens, while samples from further internal organs (i.e. liver and spleen) were negative in all animals. This study identified the brain as the most important predilection site and therefore the most appropriate tissue for T. gondii detection.
- MeSH
- Animal Structures parasitology MeSH
- Real-Time Polymerase Chain Reaction instrumentation methods MeSH
- Magnetics MeSH
- Sheep MeSH
- Toxoplasma genetics isolation & purification MeSH
- Toxoplasmosis, Animal parasitology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
