MiRNA Isolation from Plants Rich in Polysaccharides and Polyphenols.

MicroRNAs (miRNAs) are a group of 18-22-nucleotide-long noncoding RNAs, which show wide array of roles in various biological and metabolic processes in both animals and plants. They are formed endogenously and have evolutionary conserved sequences. RNA quality is extremely important for sequencing miRNAs using next-generation sequencing platforms. However, isolation and quantification of miRNAs from plant samples are often technically difficult and recovery of miRNAs from total RNA might be problematic. Degradation of RNA weakens the true miRNAs present in the sample, hence it is crucial to use a protocol that effectively retains good integrity miRNAs. In this chapter, we outline few protocols that can be used to maximize the retrieval of good-quality miRNA and total RNA to be used in miRNA analysis for plants rich in polysaccharides and polyphenols.

High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols.

Due to the presence of a diverse array of metabolites, no standard method of RNA isolation is available for plants. We noted that polysaccharide and polyphenol contents of cardamom tissues critically hinder the RNA extraction procedure. Hence, we attempted several methods for obtaining intact mRNA and small RNA from various cardamom tissues. It was found that protocols involving a combination of commercial kits and conventional CTAB (cetyl trimethylammonium bromide) methods yielded RNA with good purity, higher yield, and good integrity. The total RNA isolated through this approach was found to be amenable for transcriptome and small RNA analysis through next-generation sequencing platforms.