• Publikační typ
• abstrakt z konference 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.

• 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

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
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikro RNA analýza   genetika   MeSH
• myši MeSH
• prekurzory RNA analýza   genetika   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• stanovení celkové genové exprese metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH

The merit of RNASeq data relies heavily on correct normalization. However, most methods assume that the majority of transcripts show no differential expression between conditions. This assumption may not always be correct, especially when one condition results in overexpression. We present a new method (NormQ) to normalize the RNASeq library size, using the relative proportion observed from RT-qPCR of selected marker genes. The method was compared against the popular median-of-ratios method, using simulated and real-datasets. NormQ produced more matches to differentially expressed genes in the simulated dataset and more distribution profile matches for both simulated and real datasets.

• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

We performed a gene expression study using RT-qPCR in Staphylococcus aureus. The influence of normalization method was investigated. We confirmed that a recent standard, using more reference genes, was the best normalization strategy. The application of the most commonly used reference genes in 2011 (gyrB and 16S rRNA gene) failed.

• MeSH
• bakteriální geny MeSH
• bakteriální RNA genetika   MeSH
• DNA gyráza genetika   MeSH
• exprese genu MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• polymerázová řetězová reakce s reverzní transkripcí normy   MeSH
• referenční standardy MeSH
• RNA ribozomální 16S genetika   MeSH
• Staphylococcus aureus genetika   MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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
• analýza nákladů a výnosů MeSH
• biologické markery krev   MeSH
• cirkulující mikroRNA krev   izolace a purifikace   MeSH
• krysa rodu rattus MeSH
• kvantitativní polymerázová řetězová reakce ekonomika   přístrojové vybavení   metody   normy   MeSH
• lidé MeSH
• reagenční diagnostické soupravy normy   MeSH
• řízení kvality * MeSH
• studie proveditelnosti MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The mutual dependence of human and animal health is central to the One Health initiative as an integrated strategy for infectious disease control and management. A crucial element of the One Health includes preparation and response to influenza A virus (IAV) threats at the human-animal interface. The IAVs are characterized by extensive genetic variability, they circulate among different hosts and can establish host-specific lineages. The four main hosts are: avian, swine, human and equine, with occasional transmission to other mammalian species. The host diversity is mirrored in the range of the RT-qPCR assays for IAV detection. Different assays are recommended by the responsible health authorities for generic IAV detection in birds, swine or humans. In order to unify IAV monitoring in different hosts and apply the One Health approach, we developed a single RT-qPCR assay for universal detection of all IAVs of all subtypes, species origin and global distribution. The assay design was centred on a highly conserved region of the IAV matrix protein (MP)-segment identified by a comprehensive analysis of 99,353 sequences. The reaction parameters were effectively optimised with efficiency of 93-97% and LOD95% of approximately ten IAV templates per reaction. The assay showed high repeatability, reproducibility and robustness. The extensive in silico evaluation demonstrated high inclusivity, i.e. perfect sequence match in the primers and probe binding regions, established as 94.6% for swine, 98.2% for avian and 100% for human H3N2, pandemic H1N1, as well as other IAV strains, resulting in an overall predicted detection rate of 99% on the analysed dataset. The theoretical predictions were confirmed and extensively validated by collaboration between six veterinary or human diagnostic laboratories on a total of 1970 specimens, of which 1455 were clinical and included a diverse panel of IAV strains.

• MeSH
• chřipka lidská diagnóza   virologie   MeSH
• infekce viry z čeledi Orthomyxoviridae diagnóza   virologie   MeSH
• lidé MeSH
• nemoci prasat diagnóza   virologie   MeSH
• One Health MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• prasata MeSH
• ptačí chřipka u ptáků diagnóza   virologie   MeSH
• ptáci virologie   MeSH
• reprodukovatelnost výsledků MeSH
• virus chřipky A, podtyp H1N1 genetika   izolace a purifikace   MeSH
• virus chřipky A, podtyp H3N2 genetika   izolace a purifikace   MeSH
• virus chřipky A genetika   izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

It has been recommended that patients with leukaemias and lymphomas undergo universal screening for SARS-COV-2 using RT-qPCR before each treatment on the grounds of their high risk of experiencing severe forms of COVID-19. This raises a conflict with different recommendations which prioritise testing symptomatic patients. We found that among 56 RT-qPCR obtained in asymptomatic patients with hematologic neoplasms before chemotherapy administration, 2 (3.5%) were positive. A negative result did not exclude SARS-COV-2 infection in 1 patient (1.8%). It is unclear what the benefit of screening for SARS-COV-2 using RT-qPCR in patients with hematologic neoplasms who receive chemotherapy is.

• MeSH
• COVID-19 * diagnóza   MeSH
• hematologické nádory * diagnóza   farmakoterapie   genetika   MeSH
• leukemie * MeSH
• lidé MeSH
• SARS-CoV-2 MeSH
• senzitivita a specificita MeSH
• testování na COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

• MeSH
• bcr-abl fúzové proteiny * genetika   MeSH
• chronická myeloidní leukemie * diagnóza   farmakoterapie   genetika   MeSH
• imatinib mesylát MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• reziduální nádor genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH