Abstract:

In this paper, eyestalks of Macrobrachium nipponense were studied as the material for seeking the best extraction method of total RNA by using RNAiso Plus, RNeasy Mini Kit, Unizol with some improvements in the predecessors. The quality of the total RNA was detected and analyzed by ordinary agarose gel electrophoresis, UV spectrophotometer and RT-PCR. The results showed that the total RNA isolated by RNAiso method had mass pigments. The total RNA obtained by improved RNAiso method had been partially dissolved and got less pigments. Although less pigment inhibit the activity of RTase to some extent, it still could be used for RT-PCR. The RNeasy Mini Kit method could get rid of pigments more efficiently, which inhibit the activity of RTase, and obtain integrate total RNA but with serious gDNA contamination. The method of RNAiso or Unizol combing with RNeasy Mini Kit could get high purity total RNA without pigments and gDNA. However, the total RNA extracted by the method of Unizol combing with RNeasy Mini Kit had much better integrity than by the method of RNAiso combing with RNeasy Mini Kit. Therefore, Unizol combing with RNeasy Mini Kit should be the best method for total RNA extraction in Macrobrachium nipponense eyestalk, and the total RNA obtained by this method could be used in the subsequent molecular biological experiments such as RT-PCR, construction of cDNA library, quantitative PCR.