DescriptionFormerlyRNA Extraction Control 670.RT-qPCR Extraction Control not only enables users of a diagnostic qPCR assay to determine if there are inhibitors in the PCR assay, but also to validate the success of the extraction step, reducing the chance of obtaining a false negative result in the sample RNA.Product HighlightsSimple – streamlined protocol for straightforward validation of RNA extraction and determination of RT-qPCR assay inhibitionSensitive – control assay identifies even small effects on RNA extraction and inhibition of amplificationOptimized - control RNA has a sequence with no known homology to any organism thereby avoiding detection of sample RNASpecific – probe-based assay designed specifically for the REC control sequenceFlexible – ideal for use with a wide range of sample types, including inhibitor-rich samples like blood, urine and sputum samplesProduct DescriptionA common practice in qPCR is to add a known amount of spiked control RNA after RNA extraction, this monitors PCR inhibition but has no value as an extraction control. The ideal situation is to have the test sample and internal control undergo the same processing prior to qPCR. Meridian has developed a RT-qPCR Extraction Control, which more closely mimics the test sample, as compared to spike controls. Genetic material from the test sample and the RT-qPCR Extraction Control is simultaneously extracted by common extraction methods, with the extraction control being as sensitive to inhibition and extraction failure as the test sample.Artificial RT-qPCR Extraction Control cells are of a known concentration, containing the Internal Control RNA sequence. This sequence contains no known homology to any organism and, importantly, has minimal interference with detection of sample RNA. The RT-qPCR Extraction Control cells are spiked into the lysis buffer with the target sample, prior to RNA extraction. Control Mix, which includes primers and probe, is then added to the reaction mix before amplification. Signal derived from the Internal Control RNA confirms the success of the extraction step. RT-qPCR Extraction Control also monitors co-purification of PCR inhibitors that may cause biased or false amplification patterns.Request InfoApplicationsPathogen detectionCancer risk assessmentGene expression analysisDrug therapy efficacyBiomarker validationCopy number variation (CNV) analysisGenotypingViral loading Illustration of the extraction processREC assesses effects of extraction as well as inhibition throughout the entire workflow. REC monitors inefficient RNA extractionISOLATE II RNA Mini kit was used to extract the RNA from HeLa cells containing A) spiked control DNA and B/ REC. To show inefficient extraction, the lysis buffer and/or binding buffer substituted with PBS. The results demonstrate complete lysis (yellow line), no lysis buffer (brown), no binding buffer (grey), no lysis and no binding buffer (pink). The results illustrate that the spiked control DNA is insensitive to extraction failure, whereas REC is sensitive, with the Ct values being affected. PCR reaction inhibitionISOLATE II RNA Mini kit was used to extract the RNA from HeLa cells containing REC. Increasing concentrations of EDTA were included in the reaction to simulate increasing levels of inhibition. The results illustrate that REC is increasingly inhibited by increasing concentrations of EDTA, showing that inhibition of PCR reaction can be identified using REC.