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| High-Quality Total RNA Extraction Method from Mesenteric Adipose Tissue of Grass Carp Ctenopharyngodon idella |
| JIANG Peng1, LUO Liting2, LI Shengjie1, FAN Jiajia1, MA Dongmei1 |
1. Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China;
2. College of Animal Science & Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China |
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Abstract In this study, an improved RNA extraction method was developed from mesenteric adipose tissue of grass carp Ctenopharyngodon idella with body weight of 80—150 g by comparative analysis of multiple optimization operation steps from the conventional single-step extraction with acid guanidinium thiocyanate-phenol-chloroform. The results showed that there was lower RNA content in mesenteric adipose tissue than that in other visceral tissues, with more easily degraded in the sample pretreatment stage. It was found that the amount of adipose tissue being increased to about 30 mg was helpful to improve RNA yield and to meet the needs of relevant tests. During the lysis process using TRIzol reagent, the mechanical homogenization time prolonged to about 3 min led to significantly reduce the degree of RNA degradation. Addition of the liquid nitrogen treatment before homogenization reduced the risk of RNA degradation. The Agilent Bioanalyzer analysis revealed that the RNA integrity number (RIN) values were 8.7—9.0 for the improved RNA samples, speculating that an additional time of homogenization formed a state of sustained shear force, which promoted guanidinium thiocyanate and other components in TRIzol reagent to break through the lipid droplet barriers and then efficiently denature endogenous ribonucleases. The quality of total RNA extracted from mesenteric adipose tissue by the improved method met the requirements of high-throughput transcriptome sequencing.
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Received: 20 September 2020
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