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

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.

Development of a 3’:5’ digital PCR assay to determine horse mRNA integrity

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Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis

“…It is a simple measure of the likely integrity of the CFTR mRNA, and the relative degradation occurring at either end. Previous 3 :5 integrity assays have used oligo dT priming of cDNA in order to determine overall mRNA integrity measured from the 3 end [27,28], but we used random primers to more accurately reflect the proportion of transcript fragments that include sequence from the 5 end, and not only those that include the poly-A tail. This allowed us to produce a rough estimate of the proportion of total transcripts from which protein translation might be initiated, therefore, providing an integrity measurement with some functional significance.…”

An optimized step‐by‐step protocol for isolation of nucleus pulposus, annulus fibrosus, and end plate cells from the mouse intervertebral discs and subsequent preparation of high‐quality intact total RNA

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