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 účinky léků   genetika   růst a vývoj   fyziologie   MeSH
• chlorid sodný farmakologie   MeSH
• DNA primery genetika   MeSH
• fyziologický stres genetika   MeSH
• klonování DNA MeSH
• komplementární DNA biosyntéza   genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mitogenem aktivované proteinkinasy genetika   MeSH
• osmotický tlak účinky léků   MeSH
• regulace genové exprese u rostlin * účinky léků   MeSH
• reverzní transkripce * účinky léků   MeSH
• RNA rostlin genetika   izolace a purifikace   MeSH
• semenáček růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid sodný MeSH
• DNA primery MeSH
• komplementární DNA MeSH
• mitogenem aktivované proteinkinasy MeSH
• RNA rostlin MeSH

Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.

• MeSH
• komplementární DNA genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• rostlinné geny * MeSH
• sekvenční analýza RNA MeSH
• Silene genetika   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• komplementární DNA MeSH

• MeSH
• fluorescenční barviva metabolismus   MeSH
• komplementární DNA analýza   genetika   MeSH
• messenger RNA analýza   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí * MeSH
• referenční standardy MeSH
• reverzní transkripce * MeSH
• RNA analýza   metabolismus   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• srovnávací studie MeSH
• Názvy látek
• fluorescenční barviva MeSH
• komplementární DNA MeSH
• messenger RNA MeSH
• RNA MeSH

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

• MeSH
• Caenorhabditis elegans chemie   MeSH
• chemická frakcionace metody   MeSH
• cirkulující mikroRNA krev   izolace a purifikace   MeSH
• extracelulární vezikuly chemie   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery krev   izolace a purifikace   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• senioři MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cirkulující mikroRNA MeSH
• nádorové biomarkery MeSH

BACKGROUND: Detection of melanoma cells in peripheral blood is a promising method for monitoring haematogenous spread of melanoma cells. It enables us to detect early metastasis and to better stratify candidates for adjuvant immunotherapy. Inconsistent data on the sensitivity and clinical relevance of this method have been reported. STUDY DESIGN: We developed a multimarker real-time reverse transcription-PCR (RT-PCR) for quantification of five melanoma markers: Melan-A, gp100, MAGE-3, MIA and tyrosinase. In this prospective study, 65 patients with resected cutaneous melanoma stage IIB-III were screened. Peripheral blood samples were collected every 3 months for the following 18 months, and circulating melanoma cells were examined and compared with clinical staging results. RESULTS: Eighteen patients relapsed during the trial and showed different types of melanoma progression. All these patients experienced statistically significant tumour marker elevation in the period from 0 to 9 months before the disease progression. MAGE-3 was the most sensitive progression marker. In patients with progression, we observed three concordant positive markers in 39% of cases, two concordant positive markers in 28%, and finally one marker in 33%. CONCLUSIONS: This report describes a multiple-marker real-time RT-PCR, which is able to provide quantitative data on melanoma markers in the peripheral blood of melanoma patients. Measurement of the studied molecular markers in our hands represents a prognostic factor and a useful method for early detection of metastasis and treatment response of melanoma patients.

• MeSH
• antigeny nádorové krev   genetika   MeSH
• antigeny sacharidové asociované s nádorem krev   genetika   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• MART-1 antigen MeSH
• melanom krev   patologie   MeSH
• melanomový antigen gp100 MeSH
• membránové glykoproteiny krev   genetika   MeSH
• messenger RNA krev   genetika   MeSH
• nádorové biomarkery krev   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny krev   genetika   MeSH
• nádory kůže krev   patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• progrese nemoci MeSH
• prospektivní studie MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• staging nádorů MeSH
• tyrosinasa krev   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky kontrolované MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• antigeny sacharidové asociované s nádorem MeSH
• MAGEA3 protein, human MeSH Prohlížeč
• MART-1 antigen MeSH
• melanomový antigen gp100 MeSH
• membránové glykoproteiny MeSH
• messenger RNA MeSH
• MIA antigen, human MeSH Prohlížeč
• MLANA protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• nádorové proteiny MeSH
• PMEL protein, human MeSH Prohlížeč
• tyrosinasa MeSH

BACKGROUND: Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) are established tumour markers and the CEA pre-operative levels in serum have prognostic value. The aim of this study was to verify the usefulness of the estimation of CEA and CK20 gene expressions in tissues of colorectal cancers and their liver metastases. PATIENTS AND METHODS: Two hundred and twenty-two specimens of colorectal cancers, their liver metastases, other liver tumours and control tissues were analysed by reverse transcription combined with real-time PCR. RESULTS: The expressions of CEA and CK20 were significantly higher in tumours than in controls; there were differences between tumour types and no relationship to staging or clinical development was found. CK20 expression was inversely dependent on grading. The CEA expression in tumours did not correlate with the CEA levels in serum, but did correlate with serum tissue-specific polypeptide antigen (TPS). CONCLUSION: The measurement of CEA and CK20 gene expressions in tumours did not supply any new prognostic information, but raised the question of the mechanism releasing CEA into the blood.

• MeSH
• adenokarcinom genetika   imunologie   patologie   sekundární   MeSH
• dospělí MeSH
• exprese genu MeSH
• karcinoembryonální antigen biosyntéza   krev   genetika   MeSH
• keratin-20 MeSH
• keratiny biosyntéza   krev   genetika   MeSH
• kolorektální nádory genetika   imunologie   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA biosyntéza   genetika   MeSH
• nádorové biomarkery krev   MeSH
• nádory jater genetika   imunologie   sekundární   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• karcinoembryonální antigen MeSH
• keratin-20 MeSH
• keratiny MeSH
• KRT20 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorové biomarkery MeSH

In healthy humans, 60-70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas. Tumor biopsies from 55 patients with B cell malignancies, 5 B-lymphoid tumor cell lines, and 20 biopsies from patients with lymphadenitis were analyzed by immunohistochemistry, flow cytometry, and reverse transcription quantitative real-time PCR. Clonality was determined by immunohistochemistry in 52/53 cases, flow cytometry in 30/39 cases, and reverse transcription quantitative real-time PCR in 33/55 cases. In conclusion, immunohistochemistry was superior to flow cytometry and reverse transcription quantitative real-time PCR for clonality identification. Flow cytometry and reverse transcription quantitative real-time PCR analysis has complementary values. In a considerable number of cases tumor cells produced both kappa and lambda light chain transcripts, but only one type of light chain peptide was produced.

• Publikační typ
• časopisecké články MeSH

Chronic myeloid leukemia (CML) is characterized by the presence of BCR-ABL fusion gene resulting from the reciprocal chromosome translocation t(9;22)(q34;q 11), karyotypically detected as Ph chromosome. BCR-ABL gene was proved to play the principal role in CML pathogenesis. It is a hallmark of CML used in diagnostics and monitoring of the response to the therapy. The most sensitive method of detecting BCR-ABL aberration is RT-PCR which is able to find a single in leukemic cell between 10(6) normal leukocytes. Monitoring of BCR-ABL transcript level by quantitative RT-PCR is of the high prognostic value. High or increasing BCR-ABL transcript number signalizes bad response to treatment and a bad prognosis. On the contrary RT-PCR negativity, low level, or decreasing BCR-ABL transcript number denotes good response to treatment and good prognosis. Q-RT-PCR can detect changes in disease status several weeks or even months earlier than other methods. In 1994 the Q-RT-PCR was introduced at the Institute of Hematology and Blood Transfusion in Prague and was used for early detection of relapse after transplantation. At present it is used in all patients with CML for monitoring of response to the treatment. We have confirmed that this precise, sensitive and non-invasive method is of the principal importance for monitoring of disease status in CML patients.

• MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• chronická myeloidní leukemie diagnóza   genetika   terapie   MeSH
• genetická transkripce MeSH
• lidé MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bcr-abl fúzní proteiny MeSH

Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.

• Klíčová slova
• RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
• MeSH
• analýza jednotlivých buněk metody   normy   MeSH
• kvantitativní polymerázová řetězová reakce metody   normy   MeSH
• lidé MeSH
• reverzní transkripce genetika   MeSH
• senzitivita a specificita MeSH
• stanovení celkové genové exprese metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Recent advances allowing quantification of RNA from single cells are revolutionizing biology and medicine. Currently, almost all single-cell transcriptomic protocols rely on reverse transcription (RT). However, RT is recognized as a known source of variability, particularly with low amounts of RNA. Recently, several new reverse transcriptases (RTases) with the potential to decrease the loss of information have been developed, but knowledge of their performance is limited. METHODS: We compared the performance of 11 RTases in quantitative reverse transcription PCR (RT-qPCR) on single-cell and 100-cell bulk templates, using 2 priming strategies: a conventional mixture of random hexamers with oligo(dT)s and a reduced concentration of oligo(dT)s mimicking common single-cell RNA-sequencing protocols. Depending on their performance, 2 RTases were further tested in a high-throughput single-cell experiment. RESULTS: All tested RTases demonstrated high precision (R2 > 0.9445). The most pronounced differences were found in their ability to capture rare transcripts (0%-90% reaction positivity rate) and in their absolute reaction yield (7.3%-137.9%). RTase performance and reproducibility were compared with Z scores. The 2 best-performing enzymes were Maxima H- and SuperScript IV. The validity of the obtained results was confirmed in a follow-up single-cell model experiment. The better-performing enzyme (Maxima H-) increased the sensitivity of the single-cell experiment and improved resolution in the clustering analysis over the commonly used RTase (SuperScript II). CONCLUSIONS: Our comprehensive comparison of 11 RTases in low RNA input conditions identified 2 best-performing enzymes. Our results provide a point of reference for the improvement of current single-cell quantification protocols.

• MeSH
• analýza jednotlivých buněk MeSH
• DNA primery metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• reprodukovatelnost výsledků MeSH
• reverzní transkriptasa metabolismus   MeSH
• RNA metabolismus   MeSH
• superoxiddismutasa 1 genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA primery MeSH
• reverzní transkriptasa MeSH
• RNA MeSH
• superoxiddismutasa 1 MeSH