Extraction of low molecular weight RNA from Citrus trifolita tissues for microRNA Northern blotting and reverse transcriptase polymerase chain reaction (RTPCR)

Abstract

The study of microRNA (miRNA), a component of low molecular weight RNA (LMW RNA), has received increasing attention in recent years. A critical prerequisite in miRNA studies is acquisition of high quality LMW RNA. LMW RNA is generally obtained from total RNA or from total nucleic acids solutions. Most traditional methods for LMW RNA isolation involve many steps and chemical reagents which upon degradation may negatively affect results. We employed a simple and quick method involving trizol for total RNA extraction from citrus tissues, then generation of LMW RNA using 4M LiCl, which have been successfully utilized in studies in our laboratory. Compared with traditional methods, this method is less expensive and produced high RNA yields while avoiding the use of phenol or other toxic reagents. In addition, the entire procedure can be completed within 4 hours with many samples being processed simultaneously. Therefore, this is a practical and efficient method for LMW RNA extraction from woody fruit crops containing high levels of polysaccharide and polyphenolics. Using the extracted LMW RNA, miRNAs were successfully detected and characterized by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blotting.

Keywords: Citrus, low molecular weight RNA, trizol reagent, 4 M LiCl, microRNAs