A suitable method for isolating total RNA and preserving samples from mosquitoes
Author of the article:HUANG Wen-Yu；LU Ying；YIN Can-Can；MA Gao-Feng；LIU Xiao；SHAO Huang-Fang； LI Meng-Nan；SUN En-Tao
Author's Workplace：School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China；School of Laboratory Medicine, Wannan Medical College, Wuhu 241002, China
Key Words：mosquitoes; SDS method; modified SDS method; RNA isolation; samples preservation
Abstract：[Objectives] To determine the best methods for isolating and preserving the total RNA of mosquitoes. [Methods] The ability of three methods to isolate total RNA from mosquitoes; the modified SDS, traditional SDS and TRIzol reagent methods, were compared. The quality of total RNA obtained was evaluated with ultraviolet spectrophotometry, agarose gel electrophoresis and RT-PCR assay. In addition, mosquito RNA samples isolated by the modified SDS method were preserved under four different conditions; room-temperature, 4 ℃, -20 ℃ and -80 ℃, and the quality of the RNA preserved under each condition compared. [Results] The average concentration values of total RNA isolated by the modified SDS method, traditional SDS method and TRIzol reagent method were 1.223 3±0.374 3, 0.675 0±0.319 8 and 0.310 0±0.123 6, respectively. Analysis of variance indicates that there was a significant difference among these three methods (F=14.767, P<0.001). LSD test results suggest that the modified SDS method achieved higher concentration values of total RNA than the other two methods (P=0.005, P<0.001). The average spectrophotometric values of total RNA isolated by the modified SDS method were in the ranges 1.8-2.0 (A260/A280) and 2.0-2.2(A260/A230). The agarose gel bands of the 28S rRNA, 18S rRNA and 5S rRNA were clear, indicating a high quality of RNA devoid of polyphenols, protein and gDNA contamination. The RNA β⁃actin gene could be amplified by RT⁃PCR assay. There was no significant degradation of total RNA samples that had been frozen at -20 ℃ and -80 ℃ after one month. [Conclusion] Our data provide strong evidence that the modified SDS method is a cost-effective and convenient way to isolate high quality mosquito RNA. Freezing mosquito RNA samples at -20 ℃ and –80 ℃ can preserve their quality for up to a month.