A homemade RNA isolation reagent and its application in the extraction of fish tissue total RNA.

【Objective】 The study was done to test a homemade RNA isolation reagent,which is convenient and economical.【Method】 With TRizol Reagent as a comparison,the homemade RNA isolation reagent was used to extract total RNA of grass carp derma.The quality of the total RNA was checked with a spectrophotometer,denaturing agarose gel electrophoresis and also RT-PCR analysis.Finally,the total RNA was used to synthesize grass carp derma cDNA.Subsequently,we applied the homemade reagent to extract total RNA of zebra fish,which was used for RT-PCR to clone the full length zebra fish E2F5 gene(1 092 bp).Then an eukaryotic expression vector pcDNA3.1-flag-Zf E2F5 and a yeast expression vector pGAD GH-Zf E2F5 were constructed,and the cloned E2F5 gene was sequenced.【Result】 The total RNA of grass carp derma extracted with the homemade RNA isolation reagent was highly qualified as that with TRizol Reagent to synthesize grass carp cDNA.The total RNA of zebra fish extracted with the reagent got a even higher quality,with which the full length E2F5 gene was successfully cloned to construct pcDNA3.1-flag-Zf E2F5 and pGAD GH-Zf E2F5.【Conclusion】 The homemade RNA isolation reagent is suitable for the extraction of fish tissue total RNA,which will satisfy the needs of cDNA synthesis and gene cloning by RT-PCR.With the total RNA extracted using the homemade reagent,we successfully conducted the grass carp cDNA synthesis and zebra fish E2F5 cloning.These will be essential materials for our future studies.