High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith

Abstract

Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of b-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 lg total RNA per gram of cells was obtained with an average purity measured as A 260 /A 280 of 2.09. A titer of 10 5 cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides

“…The dilution rate was set at 0.5 h −1 , the feeding rate was maintained at 0.83 l h −1 and the working volume was 1.55 l, which made the growth rate similar to exponential growth in a shaking flask and resulted in a generation time of 2 h. Steady-state samples (OD 600 nm ~1.7) were harvested by centrifugation (6,200 g at 4 °C for 5 min) and cell pellets were frozen in liquid nitrogen. Total RNA was isolated using Trizol (Invitrogen) after grinding and was then treated with DNase I (RNase-free, NEB) to remove residual DNA52. The quality of the RNA was checked using an Agilent 2100 bioanalyser.…”

Isolation of high quality RNA from Polyporus umbellatus (Pers.) Fries

“…The other two commercial kits and improved CTAB were not efficient, which was also reported in other fungal species, i.e. Rhodosporidium toruloides (Yang et al 2010). The improved CTAB-LiCl described here efficiently eliminated most of the secondary metabolites in lotus (Nelumbo nucifera Gaertn.…”

“…These described methods often are difference because of different second metabolites contained in different fungal species (Sánchez-Rodríguez et al 2008;Yang et al 2010). On the other hand, to investigate the molecular mechanism of polysaccharide accumulation in Chuling, the intact and high quality RNA need to be obtained at first.…”

Short Communication Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues

“…In fact, CTAB-based methods have recently been used for nucleic acid isolation from polysaccharide-rich plants, and in particular, these methods have been used for RNA isolation (Thanh et al, 2009;Abbasi et al, 2010;Wang et al, 2012). Both A 260 /A 230 and A 260 /A 280 ratios were more than 2 suggesting that the RNA was of high purity and free of polysaccharide/polyphenol and protein contamination, respectively (Table 1) (Wang et al, 2005;Yang et al, 2011 Table 1. Absorbance ratios and yield of total RNA isolated from five tissues of lotus (Nelumbo nucifera) with three replications by using the selected CTAB-LiCl method.…”