An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

Abstract

High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added β-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. The modified Trizol method 2 could better extract RNA from E.senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E.senticosus Maxim.