Efficient isolation of high quality RNA from tropical palms for RNA-seq analysis

Abstract

Currently, RNA-seq as a high throughput technology is being widely applied to various species to elucidate the complexity of the transcriptome and to discover large number of novel genes. However, the technology has had poor success in elucidating the transcriptome of tropical palms, as it is difficult to isolate high quality RNA from tropical palm tissues due to their high polysaccharide and polyphenol content. Here, we developed an RNA-isolation protocol for tropical palms, the MRIP method (Methods for RNA Isolation from Palms). The integrity of the RNA molecules extracted using this protocol was determined to be of high quality by means of gel electrophoresis and Agilent 2100 Bioanalyzer microfluidic electrophoresis chip examination with a RIN (RNA Integrity Number) value of more than 9, indicating that the mRNAs were of good integrity. Subsequently the isolated RNA was used for transcription analysis without further purification. With Illumina sequencing, we obtained 54.9 million short reads and then conducted de novo assembly to gain 57,304 unigenes with an average length of 752 base pairs. Moreover, the RNA isolated with this protocol was also successfully used for real-time RT-PCR. These results suggested that the RNA isolated was suitable for Illumina RNA sequencing and quantitative real-time RT-PCR. Furthermore, this method was also successful in isolating total RNA from the leaves of various Palmaceae species.