RT-qPCR System Research Articles

Multiplex Amplification Targets for Gene Expression Analysis in a 1-Step Probe-Based RT-qPCR System

BioTechniquesVol. 61, No. 1 Sponsored Paper - Application ForumOpen AccessMultiplex amplification targets for gene expression analysis in a 1-step probe-based RT-qPCR systemSamantha Lewis & Trista SchagatSamantha LewisSearch for more papers by this author & Trista SchagatSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114437AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit OverviewThe ability to multiplex amplification targets for probe-based gene expression analysis of samples can save researchers time and money. Promega has developed an optimized set of reagents for 1-step probe-based chemistry detection, the GoTaq® Probe 1-Step RT-qPCR System, which we will show can be used for multiplex gene expression analysis with RNA samples. This master mix is also resistant to many of the common PCR inhibitors that challenge researchers.Benefits of Multiplexing:Saves money, time, and reagentsUses less sample and Master MixMaximizes instrumentation timeMethods and discussionsGene expression analysis of RNaseP, GAPDH, HPRT, and B2M was performed with human total colon RNA (Clonetech Laboratories) using the GoTaq® Probe 1-Step RT-qPCR System. An Applied Biosystems® 7500 FAST Real Time PCR System was used for amplification. Serial dilutions of RNA were run in single or 4-plex (Figure 1A-D). Cq values and amplification efficiencies were comparable for reactions in multiplex and singleplex reactions over a wide range of RNA concentrations. These results demonstrate successful multiplexing with four targets in a single reaction.Figure 1. Expression of the B2M (a), HPRT (b), GAPDH (c) and RNaseP (d) genes analyzed with a serial dilution of human total colon RNA in 4-plex or single reaction. Bars and error represent average ± standard deviation of N=4. PCR efficiencies are: 98.81% for B2M singleplex and 92.42% for B2M 4-plex, 98.22% for HPRT singleplex and 103.79% for HPRT 4-plex, 106.22% for RNaseP singleplex and 107.39% for RNaseP 4-plex, and 97.90% for GAPDH singleplex and 91.58% for GAPDH 4-plex.ConclusionThe GoTaq® Probe 1-Step RT-qPCR System can be used in qPCR studies that require multiple genes of interest to be analyzed in the same reaction well. We demonstrated that the GoTaq® Probe 1-Step RT-qPCR System can be successfully used for multiplex reactions with four targets.The benefits of this system include:Sensitive detection for earlier quantitation of low- and high-copy number targets.Resistance to a wide range of PCR inhibitors.Room temperature setups, suitable for automation and high-throughput detection.Compatible with both fast and standard qPCR cycling methodsBacked by Promega's PCR Satisfaction GuaranteeLearn more at www.promega.com/ProbeAndDyeFiguresReferencesRelatedDetails Vol. 61, No. 1 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print July 2016 Information© 2016 Future Science LtdPDF download

Canine distemper virus detection by different methods of One-Step RT-qPCR

ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

CIRCULATING EXTRACELLULAR MICRORNAS IN THE CHRONIC HYPOXIA MOUSE MODEL OF PULMONARY HYPERTENSION

s S39 apoptosis and proliferation. LncRNAs were preamplified to facilitate detection, and quantified with a commercial PCR array platform (RT2 system, Qiagen). RESULTS: Only 2 lncRNAs (ZFAS1, GAS5) were detectable in all subjects, while 63 lncRNAs were detected sporadically and 19 lncRNAs were not detectable in any subjects (based on a Cq cutoff threshold of 35). No statistically significant differences in lncRNA levels were observed between groups. The technical performance of the RT-qPCR system was confirmed by integrated reverse transcription and positive PCR controls, which revealed no evidence of reaction inhibition. The quality of the total RNA extraction was validated by the robust recovery of an exogenous spike-in control (cel-miR-39), and the detection of endogenous miRNA (miR-16, miR-126, and miR-145, without preamplification), small nuclear RNA (RN7SK), ribosomal RNA (RPLP0) and messenger RNA (ACTB, B2M) species. CONCLUSION: To the best of our knowledge, this study represents the first examination of circulating lncRNAs in PAH patients. These results suggest that low circulating levels of lncRNA may preclude their use as practical biomarkers for certain diseases. 074 CIRCULATING EXTRACELLULAR MICRORNAS IN THE CHRONIC HYPOXIA MOUSE MODEL OF PULMONARY HYPERTENSION K Schlosser, M Taha, Y Deng, DJ Stewart

RIOCIGUAT FOR CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION - INITIAL EXPERIENCE

s S39 apoptosis and proliferation. LncRNAs were preamplified to facilitate detection, and quantified with a commercial PCR array platform (RT2 system, Qiagen). RESULTS: Only 2 lncRNAs (ZFAS1, GAS5) were detectable in all subjects, while 63 lncRNAs were detected sporadically and 19 lncRNAs were not detectable in any subjects (based on a Cq cutoff threshold of 35). No statistically significant differences in lncRNA levels were observed between groups. The technical performance of the RT-qPCR system was confirmed by integrated reverse transcription and positive PCR controls, which revealed no evidence of reaction inhibition. The quality of the total RNA extraction was validated by the robust recovery of an exogenous spike-in control (cel-miR-39), and the detection of endogenous miRNA (miR-16, miR-126, and miR-145, without preamplification), small nuclear RNA (RN7SK), ribosomal RNA (RPLP0) and messenger RNA (ACTB, B2M) species. CONCLUSION: To the best of our knowledge, this study represents the first examination of circulating lncRNAs in PAH patients. These results suggest that low circulating levels of lncRNA may preclude their use as practical biomarkers for certain diseases. 074 CIRCULATING EXTRACELLULAR MICRORNAS IN THE CHRONIC HYPOXIA MOUSE MODEL OF PULMONARY HYPERTENSION K Schlosser, M Taha, Y Deng, DJ Stewart

Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs.

MicroRNAs regulate gene expression at the post-transcriptional level. Differential expression of miRNAs can potentially be used as biomarkers for early diagnosis and prediction for outcomes. Failure in validation of miRNA profiles is often caused by variations in experimental parameters. In this study, the performance of five extraction kits and three RT-qPCR systems were evaluated using BioMark high-throughput platform and the effects of different experimental parameters on circulating miRNA levels were determined. Differences in the performance of extraction kits as well as varying accuracy, sensitivity and reproducibility in qPCR systems were observed. Normalisation of RT-qPCR data to spike-in controls can reduce extraction bias. However, the extent of correlation for different qPCR systems varies in different assays. At different time points, there was no significant fold change in eight of the plasma miRNAs that we evaluated. Higher level of miRNAs was detected in plasma as compared to serum of the same cohort. In summary, we demonstrated that high-throughput RT-qPCR with pre-amplification step had increased sensitivity and can be achieved with accuracy and high reproducibility through stringent experimental controls. The information provided here is useful for planning biomarker validation studies involving circulating miRNAs.

562 Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation pathway

Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types of cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-RNAs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDAMB-231 breast cells stimulated by LPA, we found that miR-21 was one of the highest up-regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or cFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1>LPA2>>LPA6=LPA7>LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced ZEB1 and miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1siRNAs also abolished LPA-induced ZEB1 and miR-21 expression. MirVana miR-21 inhibitor, silencing LPA1 or ZEB1 totally blocked in vitro LPAinduced cell migration and invasion, and in vivo tumor bone colonization. In all cases the in vitro and in vivo breast cancer functions were rescued with miR-21 mimic. All together our results demonstrate that miR-21 controls the pro-metastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells.

Abstract 1448: Disseminated tumor cell formation promoted by lysophosphatidic acid (LPA) involves ZEB1/miR21-dependent activation pathway

Abstract Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-ARNs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDA-MB-231 breast cells stimulated by LPA, we found that miR-21 was one of the most up regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced a rapid increase in miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells reaching a plateau by two to three folds (EC50 = 0.1μM) after 45 min of stimulation. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or sFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1>LPA2>>LPA6=LPA7>LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1-siRNAs also abolished LPA-induced miR-21 expression. We showed in animal that Debio0719, another LPA1/LPA3 antagonist, inhibits implantation of disseminated tumor cells (DTC) into the bone marrow. Here, we found that the pretreatment of MDA-B02 cells with a specific blocker of miR-21 (mirVana miR-21 inhibitor) before injection of these cells in BALB/c nude mice decreased the incidence of DTC-positive animals and decreased the number DTCs at the metastatic sites. All together our results demonstrate that miR-21 controls the prometastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells. Citation Format: Debashish Sahay, Raphaël Leblanc, Johnny Ribeiro, Philippe Clézardin, Olivier Peyruchaud. Disseminated tumor cell formation promoted by lysophosphatidic acid (LPA) involves ZEB1/miR21-dependent activation pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2014-1448

A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach

BackgroundCo-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach.FindingsWe developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 102 and 104 copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR.ConclusionsThe SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.

Comparison of different RT-qPCR assays for the detection of human and bovine group A rotaviruses and characterization by sequences analysis of genes encoding VP4 and VP7 capsid proteins

The aim of this study was to compare the performance of four RT-qPCR assays for the detection of human and bovine group A rotaviruses and to characterize the positive samples by sequence analysis of VP4 and VP7 genes. RNA extracted from eight human rotavirus strains, and a panel of 33 human and 25 bovine faecal samples was subjected to different RT-qPCR detection systems. Among these assays, only RT-qPCR primers and probe systems B and C were able to detect all human rotavirus strains from cell culture solutions and faecal samples. However, the results showed that the system C was generally more sensitive by one or two logs than the other RT-qPCR assays tested. With the bovine faecal samples, the most efficient RT-qPCR systems were B and A with the detection in 100 and 92% of samples tested, respectively. Human group A rotavirus G1P[8] and bovine G6P[11] were the most frequently used strains identified in this study. A G3P[9] strain, closely related to a feline rotavirus isolated in the USA, was also discovered in a human rotavirus infection. The RT-qPCR system B was the only TaqMan assay evaluated in this study able to detect rotavirus RNA in all positive human and bovine faecal samples. Utilization of only one RT-qPCR for the detection of human and bovine group A rotaviruses and the possibility of human infection by a feline rotavirus strain.

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.

Detection and Typing of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus by Multiplex Real-Time RT-PCR

Porcine reproductive and respiratory syndrome (PRRS) causes economic losses in the pig industry worldwide, and PRRS viruses (PRRSV) are classified into the two distinct genotypes “North American (NA, type 2)” and “European (EU, type 1)”. In 2006, a highly pathogenic NA strain of PRRSV (HP-PRRSV), characterized by high fever as well as high morbidity and mortality, emerged in swine farms in China. Therefore, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay specific for HP-PRRSV was developed and combined with type 1- and type 2-specific RT-qPCR systems. Furthermore, an internal control, based on a heterologous RNA, was successfully introduced. This final multiplex PRRSV RT-qPCR, detecting and typing PRRSV, had an analytical sensitivity of less than 200 copies per µl for the type 1-assay and 20 copies per µl for the type 2- and HP assays and a high diagnostic sensitivity. A panel of reference strains and field isolates was reliably detected and samples from an animal trial with a Chinese HP-PRRS strain were used for test validation. The new multiplex PRRSV RT-qPCR system allows for the first time the highly sensitive detection and rapid differentiation of PRRSV of both genotypes as well as the direct detection of HP-PRRSV.

The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable surfaces

To demonstrate that produce rinsates used for RT-qPCR detection of foodborne viruses may cause significant PCR inhibition and propose a means to reduce its impact on sensitivity. Here, it is shown that rinsing and concentration from spinach and precut lettuce have the potential to generate RNA extracts that are inhibitory to RT-qPCRs assembled from commercial kits for the detection of norovirus GII (NoV GII), hepatitis A virus (HAV), hepatitis E virus (HEV), rotavirus (RV) and feline calicivirus (FCV) as sample process control. It is further shown that the addition of bovine serum albumin (BSA) to those reactions restored a positive signal in all cases. The effect of BSA was dependent upon the primer/probe combination. Moreover, two of the detection systems (FCV and HAV) strongly benefited from the addition of BSA even in the absence of PCR inhibitors. BSA was shown to restore positive signals in five different RT-qPCR systems that were otherwise completely inhibited by produce rinsate extracts. It is therefore suggested to consider the addition of BSA to RT-qPCRs for the detection of foodborne viruses when inhibition is observed. This study clearly demonstrates the potency of PCR inhibitors generated during routine virus concentration from produce and that it can be alleviated by the addition of BSA to the RT-qPCRs. Although used elsewhere, the addition of BSA to PCRs is not a common practice in this growing field of research.

Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR

Although uncommon in North America, Hepatitis E virus (HEV) has been identified in some industrialized countries in patients without a history of travel to HEV-endemic countries. Its presence is ubiquitous worldwide in swine populations. Zoonotic transmission of swine HEV to non human primates has been achieved experimentally and transmission of HEV after ingestion of contaminated raw or undercooked meat is well documented. In Canada, so far, no HEV outbreak has been documented but HEV genotype 3 strains have been identified in sera and faecal samples of swine origin. The objective of the present study was to determine the viral load of HEV in liver, loin, bladder, hepatic lymph node, bile, tonsil, plasma and faeces samples of 43 pigs at slaughter. Feline calicivirus (FCV) was used as sample process control to validate the RNA extraction process, as a confirmation of the absence of sample inhibitors and as an amplification control. Using FCV/HEV multiplex TaqMan RT-qPCR system, HEV RNA was detected in 14 out of the 43 animals tested. HEV was detected in lymph nodes (11/43), bladder (10/43), liver (9/43), bile (8/43), faeces (6/43), tonsils (3/43), plasma (1/43) samples from infected animals. No HEV-positive loin samples were observed. Viral loads of 10 3 to 10 7 copies/g were estimated in positive liver and bile samples.

A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA

We developed a modular real-time (rt) PCR system for absolute quantification of human nuclear (n) and mitochondrial (mt) DNA. For determination of the number of amplifiable template molecules with a minimum length required for downstream genotyping and assessment of the PCR-relevant degradation grade of the template DNA, primers yielding differently sized PCR products (nDNA: 79, 156, and 246 bp; mtDNA: 102, 143, 283, and 404 bp) and TaqMan hybridization probes were used for amplification and on-line product detection. DNase-degraded DNA served as model to demonstrate the effects of DNA fragmentation on rtPCR quantification and subsequent genotyping. Introduction of cloned internal amplification positive controls (IPCs) – generated by in vitro mutagenesis of primer-binding sites of the wild-type nDNA and mtDNA targets – enabled functionality-testing of the reaction mixture and detection of PCR inhibitors in DNA extracts, without a need for additional TaqMan probes. A hematin model was used to test the ability of the quantitative real-time (rtq) PCR system to predict the effects of inhibitors in downstream PCR-based genotyping.

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food.

To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.