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

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 (A 260 /A 280, and A 260 /A 230 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 :A 260 /A 280 ratio = 1.89 ± 0.13; A 260 /A 230 ratio = 1.7 ± 0.16) compared to the standard protocol (mean: A 260 /A 280 ratio = 1.7 ± 0.26; A 260 /A 230 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. [Display omitted] • Rat pancreas has a high level of RNase, which is one of the RNA extraction threats. • The standard TRIzol protocol provided degraded RNA of low concentration. • This study provided golden tips to obtain simple and reproducible protocol. • Improvement of RNA extraction help understand active pathways of pancreas. [ABSTRACT FROM AUTHOR]