A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (rTth) enzyme was developed to detect and quantify rotavirus A (RVA). By using rTth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.

uantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types.

Background and Objectives: We report a new real-time reverse transcription polymerase chain reaction (RT-PCR) method for quantification of TEL-AML1 transcripts. The method is based on hybridization probe (HybProbe) chemistry applied in LightCycler equipment. The study group comprised 44 successive cases of pediatric acute lymphoblastic leukemia (P-ALL).

Design and Methods: The quantitative estimation of TEL-AML1 transcripts was performed in 10 P-ALL TEL-AML1-positive patients. The PCR was performed in capillary tubes in 10 microL final volumes using two sets of primers: M1, which detects the long (L-form) and short (S-form) transcripts, and M2, specific for the L-form. The fluorescently labeled HybProbes (TEL 3FL and TEL 5LC) hybridize to the TEL region. TEL-AML1 expression was normalized relative to the levels of AML1 transcripts. Standard curves were prepared using serial dilutions of plasmids with TEL-AML1 or AML1 inserts.

Results: The sensitivity attained allowed the detection of TEL-AML1 transcripts at a 10-4 dilution of a cDNA sample from a patient at diagnosis. The within-assay coefficient of variation (CV) for TEL-AML1 was 7.0% and the between-assay CV was 13%. Levels of TEL-AML1 transcript and the TEL-AML1/AML1 ratio decreased by more than four log units (p <0.001) during or after the course of initial treatment. Most of the patients who achieved complete remission after 5-6 months of initial treatment were TEL-AML1 negative, although some positive samples with negligible amounts of TEL-AML1 transcripts were still detected.

Interpretation and Conclusions: This method has the sensitivity and reliability required to monitor the presence of minimal residual disease, and could be a powerful tool in monitoring the efficacy of the response to chemotherapy.

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.