Simplified protocol modification of TRIzol method for extraction of high-quality RNA yield from RNase-rich rat pancreas

Abstract

The extraction of a sufficient quantity of intact RNA from RNase-rich rat pancreas for estimation of gene transcript levels has been a long-standing challenge. This study provided simple modifications in the steps involved in the standard TRIzol RNA extraction method to set up a reproducible, applicable, and efficient protocol to be routinely used in the laboratory. A Nanodrop spectrophotometer was used to evaluate the RNA concentration, and purity (A260/A280, and A260/A230 absorbent ratios). Gel electrophoresis was used to assess RNA integrity. Further analysis included sequential RT-qPCR of a housekeeping gene to evaluate the optimized protocol. The current study demonstrated that the modified protocol rendered significantly more RNA concentration (mean: 635.8 ± 210.5 ng/μL) compared to the standard protocol (mean: 77.2 ± 25.9 ng/μL). Moreover, the modified protocol produced an improvement in the RNA purity (mean :A260/A280 ratio = 1.89 ± 0.13; A260/A230 ratio = 1.7 ± 0.16) compared to the standard protocol (mean: A260/A280 ratio = 1.7 ± 0.26; A260/A230 ratio = 2.5 ± 1.9). Additionally, RT-qPCR analysis revealed that the RNA extracted using the modified protocol resulted in significantly decreased (P < 0.001) Ct values (decreased by 6 Ct units) compared to the standard protocol, reflecting the good quality of the RNA. Conclusion: This study succeeded in providing golden tips to extract a high yield of good-quality RNA from RNase-rich rat pancreas for the sequential gene expression assays.