A new molecular method to assess RNA/DNA integrity with qPCR
By using paired qPCR assays that produce amplicons of different lengths from the same target, the quality of the RNA can be evaluated.
In intact mRNA the Cq's of the short and long amplicons are virtually the same, while for fragmented RNA the Cq of the longer amplicon is increased relative to that of the shorter. The TATAA DeltaAmp Quality Assays consist of short, medium and long qPCR assays that have a common reverse primer.
Three different targets are available for SYBR® or Probe based assays; Beta-2-Microglobulin (B2M), 18S rRNA (18S) and an Endogenous RNase Resistant marker (ERR).
Integrity of the mRNA has major impact on the quality of measured expression levels. This is independent of the measurement technique, may it be Next Generation Sequencing (NGS), Quantitative Real-Time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be unreliable and the whole study is likely a waste of time and money. It is therefore a common strategy to test the quality of RNA in samples before conducting large and expensive studies.
Instructions for use
The TATAA DeltaAmp Quality Assays are intended to be used to assess the quality of RNA in human samples by qPCR measurement, and consist of short, medium and long qPCR assays. The difference in Cq's of a longer and shorter assay for the same target reflects and indicates the integrity of the RNA in the sample, and can be evaluated by the score ΔAmp = CqAmplicon1 – CqAmplicon2.
As a second approach amplicon 1 could be the amplicon of an mRNA marker (such as a reference gene, for example the B2M assay) that has normal sensitivity to RNases, and amplicon 2 the amplicon of the short assay for the ERR marker. Also in this case the ΔAmp = CqAmplicon1 – CqAmplicon2 can be used as an indicator of RNA integrity.
For optimal use of these assays it is recommended to compare the ΔAmp-score of the unknown samples to that of sample(s) of known good quality.
- Biomolecular Detection and Quantification (2015B)
- The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data, Korenkova et. al.
- User Manual DeltAmp 18S Probe FAM
- User Manual DeltAmp 18S SYBR
- User Manual DeltAmp B2M Probe FAM
- User Manual DeltAmp B2M SYBR
- User Manual DeltAmp ERR Probe FAM
- User Manual DeltAmp ERR SYBR