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.
Highlights
Reverse Transcription quantitative real-time PCR (RT-qPCR) is widely used to measure relative changes in gene transcript levels in order to assess biological responses associated with disease or toxicant/drug exposure, and to validate high throughput microarray and RNA-seq data [1,2,3]
PCR reactions for Rn-3′ and Rn-5′ were performed in separate tubes using intact brain RNAs (RIN > 8.0) from five different rat strains (Wistar, Lewis, Sprague-Dawley, Long-Evans and Fischer) and the specificity of primer sets for the 3′ and 5′ amplicons was assessed by endpoint RT-PCR
Given that microfluidic-based electrophoresis systems may not be universally accessible in laboratories performing rat in vivo or in vitro toxicology studies, the development of an alternative option for rat RNA integrity assessment may contribute to improved monitoring and reporting of RNA quality controls
Summary
RT-qPCR is widely used to measure relative changes in gene transcript levels in order to assess biological responses associated with disease or toxicant/drug exposure, and to validate high throughput microarray and RNA-seq data [1,2,3]. The use of degraded RNA samples can lead to unreliable gene expression data and proper evaluation of RNA integrity is essential for reproducible results [4,5,6,7]. RIN values above 8.0 indicate intact, high quality RNA samples, between 5.0 and 8.0 moderately degraded samples, and below 5.0 degraded samples [5,6]. The use of RNA samples presenting RIN values above 5.0 is typically recommended to ensure reliable quantification of gene expression by RT-qPCR [5,6]
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