Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included

Transcription of nanos-1 in Zebrafish Embryos is not Affected by Bisphenol A: Evaluated Using Quantitative Real-Time PCR

The presence of primordial germ cells (PGCs) is crucial for proper gonad formation in zebrafish (Danio rerio). The many aspects of PGC migration that allow these cells to reach the proper location at the gonadal ridge include receptors, ligands, germ plasm components, and internal maintenance of PGCs. Any one of these factors could be affected by endocrine-disrupting chemicals (EDCs), which have been shown to alter the directed migration of these cells during early embryonic development. Based on recent research wherein the EDC bisphenol A (BPA) inhibited normal PGC migration, we have used the same dose of BPA to determine the impact of BPA on a gene central to proper germ cell migration. Zebrafish embryos were exposed to BPA, and the levels of the target gene nanos-1 were analyzed using quantitative real-time PCR (q-PCR). The target gene nanos-1 is a critically important germplasm component that allows for survival and proper migration of PGCs. The q-PCR results showed that BPA did not affect the transcription level of nanos-1 in zebrafish embryos

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

BACKGROUND: Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. METHODOLOGY/PRINCIPAL FINDINGS: Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect<or=1% mutant cells, provided that samples yielded cycle thresholds (Ct)<29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). CONCLUSIONS/SIGNIFICANCE: Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making

Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results

Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases

Background: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. Patients and methods: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. Results: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. Significance: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination

Selection of DDX5 as a novel internal control for Q-RT-PCR from microarray data using a block bootstrap re-sampling scheme

<p>Abstract</p> <p>Background</p> <p>The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.</p> <p>Results</p> <p>Sixty-six Affymetrix microarray slides using lung adenocarcinoma tissue RNAs were analyzed by a statistical re-sampling method in order to detect genes with minimal variation in gene expression. By this approach, we identified <it>DDX5 </it>as a novel internal control for Q-RT-PCR. Twenty-three genes, which were differentially expressed between adjacent normal and tumor samples, were selected and analyzed using 24 paired lung adenocarcinoma samples by Q-RT-PCR using two internal controls, <it>DDX5 </it>and <it>GAPDH</it>. The percentage correlation between Q-RT-PCR and microarray were 70% and 48% by using <it>DDX5 </it>and <it>GAPDH </it>as internal controls, respectively.</p> <p>Conclusion</p> <p>Together, these quantification strategies for Q-RT-PCR data processing procedure, which focused on minimal variation, ought to significantly facilitate internal control evaluation and selection for Q-RT-PCR when corroborating microarray data.</p

Molecular Monitoring of BCR-ABL Transcripts after Allogeneic Stem Cell Transplantation for Chronic Myeloid Leukemia

AbstractThe monitoring of minimal residual disease (MRD) through low sensitivity real-time (RT) polymerase chain reaction (PCR) analysis of BCR-ABL transcripts allows early detection of chronic myeloid leukemia (CML) relapse after allogeneic hematopoietic stem cell transplantation (HSCT). The introduction of more sensitive techniques, such as RT quantitative (Q)-PCR, may lead to an overestimation of the risk of CML relapse. In this study, we reviewed the results of peripheral blood RT Q-PCR in CML patients who underwent allogeneic HSCT from 1983 to 2007. In our laboratory, RT Q-PCR analysis was routinely performed since 2002. Eighty-seven of 189 patients had available RT Q-PCR data; 63 patients had at least 3 RT Q-PCR analyses assessable. Fifty-two of 63 patients (83%) had, at least once, detectable transcript levels, but with an BCR-ABL/ABL ratio <.1% defined as .1% confirmed by the finding of Ph+ cells in bone marrow. No patients with persistent undetectable transcripts relapsed (P = .19). Relapse did not correlate with the number of occurrences o

Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR

A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed

The Prevalence of the Q-fever Agent Coxiella burnetii in Ticks Collected from an Animal Shelter in Southeast Georgia

Author\u27s abstract: Q-fever is a zoonosis caused by a worldwide-distributed bacterium Coxiella burnetii. Ticks are vectors of the Q-fever agent but play a secondary role in transmission because the agent is also transmitted via aerosols. Most Q-fever studies have focused on farm animals but not ticks collected from dogs in animal shelters. In order to detect the Q-fever agent in these ticks, a nested PCR technique targeting the 16S rDNA of Coxiella burnetii was used. A collection of 450 ticks from the animal shelter were screened via nested PCR and 144 (32%) were positives. The positive PCR products were also confirmed by DNA sequencing. This is the first report of the prevalence of the Q-fever agent in ticks from an animal shelter. The results are significant to public health. Highly infected ticks in animal shelters may transmit the Q-fever agent to humans via its feces, excretion, or by biting

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides

Copyright @ 2012 John Wiley & SonsThe miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C-ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor-stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C-ALCL and BID. The up-regulation of miR-155, miR-27b, miR-30c and miR-29b in C-ALCL was validated by miRNA-Q-PCR on independent study groups. Additionally, the miRNA expression profiles of C-ALCL were compared with those of tumor-stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA-Q-PCR demonstrated statistically significantly differential expression of miR-155, miR-27b, miR-93, miR-29b and miR-92a between tumor-stage MF and C-ALCL. This study, the first describing the miRNA expression profile of C-ALCL, reveals differences with tumor-stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.This work was funded by grants from Netherlands Organization for Scientific Research (NWO) (MHV) and the Fondation Rene´ Touraine (MvK), and grants from the Leukaemia and Lymphoma Research (EB) and the Julian Starmer-Smith Memorial Fund (CHL)