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. 2015 Sep 26:6:4-12.
doi: 10.1016/j.bdq.2015.09.002. eCollection 2016 Jan.

Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity

J Björkman  1 , D Švec  2 , E Lott  1 , M Kubista  2 , R Sjöback  1
Affiliations

Differential amplicons (ΔAmp)-a new molecular method to assess RNA integrity

J Björkman et al. Biomol Detect Quantif. .

Abstract

Integrity of the mRNA in clinical samples has major impact on the quality of measured expression levels. This is independent of the measurement technique being next generation sequencing (NGS), Quantitative real-time PCR (qPCR) or microarray profiling. If mRNA is highly degraded or damaged, measured data will be very unreliable and the whole study is likely a waste of time and money. It is therefore common strategy to test the quality of RNA in samples before conducting large and costly studies. Most methods today to assess the quality of RNA are ignorant to the nature of the RNA and, therefore, reflect the integrity of ribosomal RNA, which is the dominant species, rather than of mRNAs, microRNAs and long non-coding RNAs, which usually are the species of interest. Here, we present a novel molecular approach to assess the quality of the targeted RNA species by measuring the differential amplification (ΔAmp) of an Endogenous RNase Resistant (ERR) marker relative to a reference gene, optionally combined with the measurement of two amplicons of different lengths. The combination reveals any mRNA degradation caused by ribonucleases as well as physical, chemical or UV damage. ΔAmp has superior sensitivity to common microfluidic electrophoretic methods, senses the integrity of the actual targeted RNA species, and allows for a smoother and more cost efficient workflow.

Keywords: DAmp; Differential amplicons; Endogenous RNase resistant marker; RNA integrity; RNA quality; ΔAmp.

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Figures

Fig. 1
Fig. 1
RNase I treated HeLa cells. Cq and RQI plotted against [RNase I] (A). ΔΔAmpERR(RNase) = ΔAmpERR ([RNase]) − ΔERR ([RNase] = 0) and RQI plotted against [RNase I] (B).
Fig. 2
Fig. 2
Room temperature incubated frozen mouse liver tissue. Cq and RQI plotted against time in room temperature for tissue powder (A) and pieces (B). ΔΔAmpERR(t) = ΔAmpERR(t)- ΔAmpERR (t = 0) and RQI plotted against time in room temperature for tissue powder and pieces (C).
Fig. 3
Fig. 3
Purified human RNA incubated at 95 °C. RQI plotted against incubation time and gel picture of all time points.
Fig. 4
Fig. 4
Purified human RNA incubated at 95 °C. RQI plotted against ΔΔAmpERR for B2 M (A), ERR marker (B) and 18S (C). Incubation time plotted against ΔΔAmpERR for B2 M (D), ERR marker (E) and 18S (F).
Fig. 5
Fig. 5
Purified human RNA exposed to UV radiation. RQI plotted against incubation time and gel picture of all time points.
Fig. 6
Fig. 6
Purified human RNA exposed to UV radiation. RQI plotted against ΔΔAmpERR for B2 M (A), ERR marker (B) and 18S (C). Exposure time plotted against ΔΔAmpERR for B2 M (D), ERR marker (E) and 18S (F).
Fig. 7
Fig. 7
RQI and ΔΔAmpX–Y for 18S rRNA as function of the time exposed to formalin.
See this image and copyright information in PMC

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