Easy monitoring and validation of RNA extraction protocols
Detection of real-time PCR inhibition
Minimal interference with sample detection
Ideal for blood, urine and sputum samples
Specially designed for real-time PCR assays
REC is suitable for use with commercially available silica-membrane RNA extraction kits and CHELEX matrices and has been tested on a wide range of real-time PCR platforms including ABI-7500, LightCycler 480®, RotorGene-Q™ and MX3005P®. To fit in with existing protocols, REC uses Quasar® 670 dye, a performance-optimized fluorophore for multiplex real-time PCR.
A common practice in real-time PCR is to add a known amount of “spiked” control DNA after RNA extraction. This monitors PCR inhibition but has no value as an extraction control or to show efficiency of reverse transcription. The ideal situation is to have the test sample and internal control undergo the same processing prior to real-time PCR (Fig. 1). Bioline have specially developed a RNA Extraction Control (REC), which more closely mimics the test sample, as compared to spike controls. The genetic material from the test sample and the REC is simultaneously extracted by common extraction methods, with the extraction control being as sensitive to inhibition and extraction failure as the test sample.
The REC consists of artificial cells 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 REC cells are spiked into lysis buffer with the target sample, prior to DNA extraction. Control Mix (primers and probe) is then added to the reaction mix before amplification. Signal derived from the Internal Control DNA confirms the success of the extraction step (Fig. 2). REC also monitors co-purification of PCR inhibitors (Fig. 3) that may cause biased or false amplification patterns.
REC offers the quality control assurance required for the entire workflow from sample extraction, reverse transcription/cDNA synthesis to real-time amplification and analysis.