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. 2018 Mar 16:15:18-23.
doi: 10.1016/j.bdq.2018.02.001. eCollection 2018 May.

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples

Bhaja K Padhi  1 , Manjeet Singh  1 , Marianela Rosales  1 , Guillaume Pelletier  1 , Sabit Cakmak  2
Affiliations

A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples

Bhaja K Padhi et al. Biomol Detect Quantif. .

Abstract

Reverse Transcription quantitative real-time PCR (RT-qPCR) is applied to quantify gene transcript levels in a wide range of investigations. Proper assessment of RNA integrity is essential for reliable assessment of gene expression levels, as RNA molecules are acutely vulnerable to degradation. However, RNA quality control measures are still infrequently reported in rat toxicological studies, which impede proper evaluation of gene expression data reliability. The high operational cost of microfluidic capillary electrophoresis systems along with paucity of alternative methods for the quantitative assessment of rat RNA integrity constitute potential hurdles to the systematic implementation and reporting of RNA integrity assessment in rat studies. This manuscript describes the adaptation of an alternative RT-qPCR-based 3':5' assay as an additional option for the quantitative assessment of rat RNA integrity. Two PCR primer sets were designed on the 3' and 5' regions of a rat housekeeping gene to evaluate RNA integrity by measuring the relative expression (3':5' ratio) of these amplicons. The 3':5' ratios were then compared to Agilent Bioanalyzer's RNA integrity number (RIN) for a wide range of RNA samples originating from different tissues, cultured cell lines and rat strains that were prepared freshly, stored for years at -80 °C, purchased commercially or intentionally degraded. The 3':5' ratios and RIN values presented similar assessment of RNA integrity status from intact to heavily degraded samples. Based on the LOWESS regression of this large comparison dataset, 3':5' ratio threshold criteria equivalent to RIN cut-off values can be proposed for the selection of RNA samples for RT-qPCR analyses. This qPCR-based assay is easy to implement, cost-effective, and provides a reliable quantification of RNA integrity to assist in the selection of rat RNA samples suitable for downstream RT-qPCR gene expression analyses.

Keywords: Gene expression; Housekeeping gene; Pgk1; RNA integrity; Rat; Real-time quantitative PCR.

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Figures

Fig. 1
Fig. 1
Development of the 3′:5′ assay. (a) Schematic representation of rat Pgk1 mRNA showing the locations of the 5′ and 3′ amplicons. Exon boxes are numbered and their lengths in bp are indicated below. Three RNA samples from C6 cells (b–c) and PND 14 hippocampi (d–e) were heat-degraded (for 0, 5, 10 and 20 min at 90 °C) and then analyzed using the Agilent Bioanalyzer system and the proposed rat 3′:5′ assay. The intensities of 28S rRNA bands on Bioanalyzer’s electropherograms gradually decreased to completely disappear after 20 min of heat treatment (c and e). The 3′:5′ ratios gradually increased with RNA degradation, reaching maximal values of up to 71.2 (b) and 49.9 (d).
Fig. 2
Fig. 2
Comparison of 3′:5′ ratios and RIN values for a panel of 99 rat RNA samples. LOWESS regression shows a clear association between 3′:5′ ratios and RIN values for the RNA samples assessed (listed in Tables S1 and S2).
Fig. 3
Fig. 3
Measurement of Tacc2 relative expression in proliferating (n = 3) and differentiating (n = 3) PC12 cells using intact (0 min heat), moderately degraded (5 min heat) or degraded (10 min heat) RNA samples. Average RIN and 3′:5′ ratio values are provided below each heat-treatment condition. Error bars represent standard error of the mean and * indicates a statistically significant difference (p < 0.05) according to two-tailed Student’s t-test.
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