... Nested PCR 35 -- 3.4. Multiplex PCR 36 -- 3.5. Differential PCR 36 -- 3.6. Competitive PCR -- 3.7. ... ... Methods for the isolation of RNA suitable for RT-PCR 91 -- XIII -- Method 1: Acid guanidinium thiocyanate-phenol-chloroform ... ... Reverse transcription/PCR (RT-PCR) -- 9.1. Setting up an RT-PCR -- 9.2. ... ... Selective RT-PCR -- 99 -- 100 -- 102 -- Method 1: RNase-free DNase treatment of RNA samples 103 -- Method ... ... Quantification of PCR-products 201 -- 17. ...
271 s. : il.
- MeSH
- Clinical Laboratory Techniques MeSH
- Molecular Biology MeSH
- Polymerase Chain Reaction MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biologické vědy
- NML Fields
- biologie
RNA viruses are pathogenic agents of many serious infectious diseases affecting humans and animals. The detection of pathogenic RNA viruses is based on modern molecular methods, of which the most widely used methods are the reverse transcription polymerase chain reaction (RT-PCR) and the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). All steps of RT-PCR and qRT-PCR should be strictly controlled to ensure the validity of obtained results. False-negative results may be caused not only by inhibition of RT or/and PCR steps but also by failure of the nucleic acid extraction step, particularly in the case of viral RNA extraction. The control of nucleic acid extraction generally involves the utilization of a non-pathogenic virus (process control virus) of similar structural properties to those of the target virus. Although in clinical samples the use of such process control virus is only recommended, in other kinds of settings such as food matrices its use is necessary. Currently, several different process control viruses are used for these purposes. Process control viruses can also be constructed artificially using technology for production of MS2 phage-like particles, which have many advantages in comparison with other used controls and are especially suited for controlling the detection and quantification of certain types of RNA viruses. The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Nevertheless, the practical use of MS2 phage-like particles in routine diagnostics is relatively uncommon. The current situation with regard to the use of MS2 phage-like particles as process control viruses in detection of RNA viruses and different methods of their construction, purification and use are summarized and discussed in this review.
- Keywords
- process control virus,
- MeSH
- RNA Virus Infections * diagnosis MeSH
- Levivirus * isolation & purification MeSH
- Humans MeSH
- Reverse Transcriptase Polymerase Chain Reaction * methods MeSH
- RNA, Viral genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
BACKGROUND: Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in tissues of the most important cereals (wheat, barley and oats). Titre of Barley yellow dwarf virus (BYDV) was determined in oats using relative quantification with different reference genes and absolute quantification, and the results were compared. RESULTS: The expression of seven potential reference genes was evaluated in tissues of 180 healthy, physiologically stressed and virus-infected cereal plants. These genes were tested by RT-qPCR and ranked according to the stability of their expression using three different methods (two-way ANOVA, GeNorm and NormFinder tools). In most cases, the expression of all genes did not depend on abiotic stress conditions or virus infections. All the genes showed significant differences in expression among plant species. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUBB) and 18S ribosomal RNA (18S rRNA) always ranked as the three most stable genes. On the other hand, elongation factor-1 alpha (EF1A), eukaryotic initiation factor 4a (EIF4A), and 28S ribosomal RNA (28S rRNA) for barley and oat samples; and alpha-tubulin (TUBA) for wheat samples were consistently ranked as the less reliable controls.The BYDV titre was determined in two oat varieties by RT-qPCR using three different quantification approaches. There were no significant differences between the absolute and relative quantifications, or between quantification using GAPDH + TUBB + TUBA +18S rRNA and EF1A + EIF4A + 28S rRNA. However, there were discrepancies between the results of individual assays. CONCLUSIONS: The geometric average of GAPDH, 18S rRNA and TUBB is suitable for normalisation of BYDV quantification in barley tissues. For wheat and oat samples, a combination of four genes is necessary: GAPDH, 18S rRNA, TUBB and EIF4A for wheat; and GAPDH, 18S rRNA, TUBB and TUBA for oat is recommended.
- MeSH
- Edible Grain genetics virology MeSH
- Luteovirus physiology MeSH
- Reverse Transcriptase Polymerase Chain Reaction methods MeSH
- Reproducibility of Results MeSH
- RNA, Ribosomal, 18S analysis genetics MeSH
- Genes, Plant genetics MeSH
- Plant Proteins genetics metabolism MeSH
- Gene Expression Profiling methods MeSH
- Viral Load MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Standard screening of melanoma patients is a useful tool for predicting outcome of patients, however, an instant methodology for exact detection of subclinical disease or monitoring treatment response is under investigation. Detection of circulating melanoma cells is, therefore, a possible novel promising staging method. However, inconsistent data on method sensitivity and on the predicted patient outcome has been shown repeatedly. Recently, a multimarker real-time RT-PCR methodology for quantification of five melanoma markers Melan-A, gp 100, MAGE-3, MIA and tyrosinase was described by our group. In the current prospective trial, blood specimens of 65 patients with AJCC stage IIB-III cutaneous melanoma after surgery were periodically examined. In the above group, 27 % of subjects relapsed during the study. Prior to the disease progression we could observe a statistically significant tumor marker elevation in previous 0 to 9 months in all patients with clinical relapse. MAGE-3 became the most sensitive progression marker. During progression, three concordant positive markers were seen in 39 % of patients, followed by two concordant positive markers in 28 % and 1 marker in 33 %. This study supports the use of a multimarker real-time RT-PCR as a disease progression predictor. The dynamic assessment of serially obtained blood specimens represents a useful method for early metastasis detection and treatment response of melanoma patients.
- MeSH
- Early Diagnosis MeSH
- Adult MeSH
- Eukaryotic Initiation Factor-3 genetics MeSH
- Financing, Organized MeSH
- Genetic Markers MeSH
- Genetic Testing methods MeSH
- Middle Aged MeSH
- Humans MeSH
- Melanoma diagnosis genetics MeSH
- Cell Line, Tumor MeSH
- Skin Neoplasms diagnosis genetics MeSH
- Reverse Transcriptase Polymerase Chain Reaction MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Controlled Clinical Trial MeSH
MicroRNAs are a class of small non-coding RNAs that serve as important regulators of gene expression at the posttranscriptional level. They are stable in body fluids and pose great potential to serve as biomarkers. Here, we present a highly specific, sensitive and cost-effective system to quantify miRNA expression based on two-step RT-qPCR with SYBR-green detection chemistry called Two-tailed RT-qPCR. It takes advantage of novel, target-specific primers for reverse transcription composed of two hemiprobes complementary to two different parts of the targeted miRNA, connected by a hairpin structure. The introduction of a second probe ensures high sensitivity and enables discrimination of highly homologous miRNAs irrespectively of the position of the mismatched nucleotide. Two-tailed RT-qPCR has a dynamic range of seven logs and a sensitivity sufficient to detect down to ten target miRNA molecules. It is capable to capture the full isomiR repertoire, leading to accurate representation of the complete miRNA content in a sample. The reverse transcription step can be multiplexed and the miRNA profiles measured with Two-tailed RT-qPCR show excellent correlation with the industry standard TaqMan miRNA assays (r2 = 0.985). Moreover, Two-tailed RT-qPCR allows for rapid testing with a total analysis time of less than 2.5 hours.
- MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- MicroRNAs analysis genetics MeSH
- Mice MeSH
- RNA Precursors analysis genetics MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Gene Expression Profiling methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3´-end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8-month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected.
- Keywords
- Rat slow soleus muscle, Rat fast EDL muscle, Myosin heavy chain isoforms, Real time RT-PCR,
- MeSH
- DNA Primers MeSH
- Financing, Organized MeSH
- Muscle, Skeletal chemistry MeSH
- Rats MeSH
- RNA, Messenger analysis MeSH
- Polymerase Chain Reaction MeSH
- Rats, Inbred Lew MeSH
- Protein Isoforms MeSH
- Muscle Fibers, Slow-Twitch chemistry MeSH
- Muscle Fibers, Fast-Twitch chemistry MeSH
- Myosin Heavy Chains genetics MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
Metoda polymerázové řetězové reakce v reálném čase (RQ-PCR) představuje v současnosti jednu z nejcitlivějších kvantitativních metod stanovení chimérismu po alogenní transplantaci krvetvorných buněk. Tato metoda je minimálně desetkrát citlivější než běžně používaná metoda fragmentační analýzy (FA) s fluorescenční detekcí. Aplikace metody umožňující stanovení mikrochimérismu, tj. smíšeného chimérismu (SC) na hladině menší než 1 %, dovoluje zachycení relapsu onemocnění signifikantně dříve a ve větším počtu případů, než je tomu u méně senzitivních metod. U pacientů s náhlým znovuobjevením autologní krvetvorby, odhalilo použití RQ-PCR SC také u předchozích odběrů, pomocí FAjiž klasifikovaných jako chimérismus kompletní (KC). Rovněž retrospektivní RQ-PCR analýza vzorků s pozitivní minimální zbytkovou chorobou, dle FA klasifikovaných jako KC, opakovaně potvrdila přítomnost autologní krvetvorby na hladině menší než 1 %. Použití metody stanovení chimérismu s lepší mezí detekce tedy umožňuje dřívější klinickou intervenci, avšak absolutní význam chimérismu i mikrochimérismu musí být dále hodnocen.
Real time polymerase chain reaction (RQ-PCR) represents one of the most sensitive methods for quantitative analysis of chimerism. This method is at least ten times more sensitive than commonly performed method of fragment analysis (FA) with fluorescence detection. Application of method that enables detection of microchimerism (mixed chimerism under 1%), can recognize relaps significantly sooner and in more cases than by using less sensitive methods. Performance of RQ-PCR within patients with sudden recurrence of autologous haematopoiesis showed microchimerism also in previous samples, formerly judged according to FA as complete donor chimerism (CDC). Furthermore, retrospective analysis of CDC samples from patient with positive residual disease repeatedly revealed microchimerism. Performance of more sensitive method can significantly contribute to early clinical intervention, nevertheless the predictable value of mixed chimerism and especially microchimerism must be further investigated.
BACKGROUND: MicroRNAs (miRNAs) are new generation biomarkers used in oncology, cardiology, metabolic syndrome, obesity or in neurology. miRNAs are short non-coding RNA molecules that regulate gene expression in eukaryotes. AIM: To compare a new commercial method for establishing miRNA (imunoassay) with a commercial kit RT qPCR. METHODS: RNA was isolated from whole blood samples obtained from four healthy volunteers. The isolates were liquated and miRNA-93-5p and miRNA-23a-3p were measured independently with commercial hsa-miR-93-5p miREIA and hsa-miR-23a-3p miREIA, and commercial RT-qPCR kits. RESULTS: Both miRNAs had good analytical characteristics, very good correlation with RT qPCR. The results between immunoassay and RT qPCR did not statistically differ. A method based on ELISA was faster (2 h with ELISA vs. 3 h with qPCR) and had lower CV then a method based on RT qPCR (see more text). CONCLUSION: MicroRNAs from blood or derived fractions are particularly interesting candidates for routine laboratory applications. The immunoassay can be performed on any device that processes the ELISA plates and is therefore available in almost every laboratory.
- MeSH
- Biomarkers blood MeSH
- Enzyme-Linked Immunosorbent Assay methods MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Humans MeSH
- MicroRNAs blood genetics MeSH
- Pilot Projects MeSH
- Reagent Kits, Diagnostic * MeSH
- Reproducibility of Results MeSH
- Gene Expression Profiling MeSH
- Healthy Volunteers MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Although it is generally accepted that signal transduction in plant mitogen-activated protein kinase signaling cascades is regulated via rapid posttranslational modifications, there are also several compelling examples of swift stress induced transcriptional activation of plant MAP kinase genes. A possible function of these fast and transient events is to compensate for protein losses caused by degradation of phosphorylated MAP kinases within stimulated pathways. Nevertheless, there is still need for additional evidence to precisely describe the regulatory role of plant MAP kinase transcriptional dynamics, especially in the context of whole stress stimulated pathways including also other signaling molecules and transcription factors. During the last two decades a reverse transcription quantitative real-time PCR became a golden choice for the accurate and fast quantification of the gene expression and gene expression dynamic. In here, we provide a robust, cost-effective SYBR Green-based RT-qPCR protocol that is suitable for the quantification of stress induced plant MAP kinase transcriptional dynamics in various plant species.
- MeSH
- Arabidopsis drug effects genetics growth & development physiology MeSH
- Sodium Chloride pharmacology MeSH
- DNA Primers genetics MeSH
- Stress, Physiological genetics MeSH
- Cloning, Molecular MeSH
- DNA, Complementary biosynthesis genetics MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Mitogen-Activated Protein Kinases genetics MeSH
- Osmotic Pressure drug effects MeSH
- Gene Expression Regulation, Plant * drug effects MeSH
- Reverse Transcription * drug effects MeSH
- RNA, Plant genetics isolation & purification MeSH
- Seedlings growth & development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
