科研之友
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摘要
An efficient RNA-extraction method is crucial for transcriptomic studies of ecologically important organisms like copepods. In this study, we used the copepod Acartia hudsonica as a test species to evaluate existing methods, and formulated several improved protocols to consistently and efficiently isolate high-quality RNA from both pooled and individual copepod samples. Our protocols recommend copepod preservation in a phenol/guanidine thiocyanate reagent followed by bead-beating or micropestle homogenization. To obtain high-quality RNA from pooled samples, one initial chloroform extraction followed by multiple phenol/chloroform extractions are needed before the raw RNA samples are further purified with the Qiagen RNeasy Mini Kit. When analyzing individual samples, one chloroform extraction followed by purification using the Zymo Research Direct-zol (TM) MiniPrep Kit gives the best results. Using these protocols, we isolated RNA from pooled and individual A. hudsonica samples with consistent recovery rate (individual samples: 95 +/- 12 ng/male; 272 +/- 57 ng/female). The cDNAs synthesized using these RNAs were proven to be of high quality by successful amplification of transcripts including the %26gt;7 kb alpha subunit of the voltage-gated sodium channel gene (SCG). For individual A. hudsonica samples, our protocols yielded significantly higher success rates (95%, n = 456) in reverse transcription quantitative PCR of SCG compared to existing methods (0-85%, n = 150). Applications of these protocols to nine other copepod species from seven families have led us to achieve high-quality RNAs and cDNAs from pooled and/or individual samples. As a result of testing the protocols, we obtained cDNA sequences of various genes in ten copepod species, some of which are the first record in copepods. Our protocols can easily be adopted by laboratories to facilitate molecular work on copepods.
- 出版日期2013-8
- 单位中国海洋大学
