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Description
Formerly RNA 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 Highlights
Simple – streamlined protocol for straightforward validation of RNA extraction and determination of RT-qPCR assay inhibition
Sensitive – control assay identifies even small effects on RNA extraction and inhibition of amplification
Optimized - control RNA has a sequence with no known homology to any organism thereby avoiding detection of sample RNA
Specific – probe-based assay designed specifically for the REC control sequence
Flexible – ideal for use with a wide range of sample types, including inhibitor-rich samples like blood, urine and sputum samples
Product Description
A 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.
Applications
Pathogen detection
Cancer risk assessment
Gene expression analysis
Drug therapy efficacy
Biomarker validation
Copy number variation (CNV) analysis
Genotyping
Viral loading
Illustration of the extraction process
REC assesses effects of extraction as well as inhibition throughout the entire workflow.
REC monitors inefficient RNA extraction
ISOLATE 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 inhibition
ISOLATE 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.
RT-qPCR Extraction Control Red is suitable for use with commercially available silica-membrane RNA extraction kits and CHELEX matrices and has been tested on a wide range of qPCR platforms including ABI-7500, LightCycler 480®, RotorGene-Q™, Mic and MX3005P®.
RT-qPCR Extraction Control Red uses Quasar® 670 and is also available with Cal Fluor® Orange 560 to fit in with existing protocols. CAL Fluor and Quasar dyes are performance-optimized fluorophores for multiplex qPCR.
All kit components should be stored at -80°C upon receipt. When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date on the outer box label.
We recommend to use the probe based detection methods for quantification of DNA and RNA extraction controls. Also, the REC kits have been designed for one-step RT-qPCR. But, it would be possible to use other PCR applications for detection and quantification of the extraction controls. Also possible are two-step PCR, SYBR® based or even endpoint PCR. If you would like to use REC with a separated reverse transcription it would be necessary to use the provided primer Control Mix also for reverse transcription reaction. If you are using SYBR® based detection methods, it is possible in singleplex only. Otherwise, the Ct value will be influenced by both the amplification of the target and extraction control.
We recommend to use a one-step RT-qPCR setup for detection of the RNA extraction control, but it would be possible to perform a two-step qPCR with separate reverse transcription. It would be necessary for this to use the provided primer control mix also for reverse transcription as hexamer or oligo dT would not give consistent results. The customer needs to optimize the amount of primer control-mix for reverse transcription.
The DNA and RNA extraction controls are suitable with any kinds of extraction method. It may be combined with e.g. phenol, column or magnetic bead based extraction methods and it is also suitable with automated extraction. The genetic material from the test sample and DEC or REC are simultaneously extracted with any common extraction method.
The benefit of the artificial DEC or REC cells in evaluation of the extraction process is the possibility to validate sample extraction process. Signal derived from the Internal Control confirms the success of the extraction step and monitors co-purification of PCR inhibitors that may cause biased or false amplification patterns. Internal control or spike in samples do not monitor the whole extraction process, especially not the lysis step. Also with quantification of internal genes it is not possible to distinguish for instance between low extraction efficiency and degraded samples.
