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- Title
A suitable method for isolating total RNA and preserving samples from mosquitoes.
- Authors
HUANG Wen-Yu; LU Ying; YIN Can-Can; MA Gao-Feng; LIU Xiao; SHAO Huang-Fang; LI Meng-Nan; SUN En-Tao
- 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.
- Publication
Chinese Journal of Applied Entomology, 2019, Vol 56, Issue 6, p1430
- ISSN
2095-1353
- Publication type
Article
- DOI
10.7679/j.issn.20951353.2019.156