关键词: 凡纳滨对虾, 总RNA完整性, 定量PCR, 3':5'分析, 隐裂

Abstract: The denaturing agarose gel electrophoresis and ethidium bromide staining is the most common method used to assess the integrity of total RNA. The 28S rRNA band is approximately twice as intense as the 18S rRNA band after running an intact total RNA sample on a denaturing gel. If the 28S:18S rRNA ratio is less than 2, it is likely that the RNA sample suffered degradation during preparation. The less the ratio is, the more is the degradation. In order to check whether it works for agarose gel analysis to assess the integrity of total RNA extracted from Penaeus vannamei, the integrity of rRNA and mRNA was assessed using two different methods, gel electrophoresis and 3':5' assay in this paper. Total RNA was extracted from Penaeus vannamei with TRIzol reagent and treated by DNase I. A very low value of 28S:18S rRNA ratio, far less than 2, was observed using agarose gel analysis, which suggested the degradation of total RNA sample during preparation. However, using the same total RNA as template, two genes of about 1 100 bp were successfully amplified by reverse transcription PCR. Furthermore, a 3':5' assay was carried out based on the same total RNA. The 3':5' assay was independent of the rRNA integrity and could assess the mRNA integrity directly. A reverse transcription reaction was primed using oligo (dT), and a real time quantitative PCR assay was used to quantitate the levels of 3' and 5' target sequences from ACT and eEF1A mRNA. The 3':5' ratio of ACT and eEF1A mRNA were 2.79 and 1.53, respectively, which indicated the high integrity of mRNA. So gel electrophoresis of RNA underestimated the integrity of total RNA. The reliable assessment of the integrity of total RNA would need the 3':5' assay.

Key words: Penaeus vannamei, total RNA integrity, real time PCR, 3':5' assay, hidden break