Isolation of total RNA from hard bamboo tissue rich in polyphenols and polysaccharides for gene expression studies

Abstract

RNA isolation from hard and woody internodal bamboo (Bambusa balcooa) tissue is very difficult due to the presence of secondary metabolites, polysaccharides, and polyphenolics. These compounds often co-precipitate with isolated RNA and hinder downstream applications. We have developed an efficient, cost effective and reproducible RNA isolation method from hard tissue of bamboo internode. This protocol includes an additional organic solvent refinement steps to remove endogenous phenolic compounds and acidic phenol (pH 4.2) to critically stabilize RNA in extraction buffer. In addition to these, two 2M Lithium chloride washing steps were introduced to eliminate DNA and polysaccharides contamination. The RNA isolated from the present protocol was found to be superior, when compared to total RNA extracted by other available protocols. The A260/A280 absorption ratio of the isolated RNA was found ranging between 1.89-1.97. The integrity of 28S and 18S rRNA was highly satisfactory when analyzed in agarose denaturing gel. RNA was further used for RT PCR, northern hybridization, cDNA library and subtractive hybridization without any further refinement.

First proteome study of sporadic flowering in bamboo species (Bambusa vulgaris and Dendrocalamus manipureanus) reveal the boom is associated with stress and mobile genetic elements

“…A piecemeal approach merging genomics, transcriptomics and proteomics can provide insights into the bamboo's death phenomenon succeeding flowering boom. Nonetheless, isolating biomolecules from bamboo tissues is often challenging because of the presence of secondary metabolites, polysaccharides and polyphenolics (Rai et al, 2010). Bamboo tissues are highly pigmented (Fig.…”

Identification of programmed cell death related genes in bamboo

“…Total RNA was isolated using the internodal tissue (5th) of control and test samples of B. arundinacea respectively using a protocol reported by Rai et al (2010), treated with DNaseI (Cat# M6101, Promega, Madison, WI, USA) and purified subsequently. The DNA-free RNA was quantified spectrophotometrically (NanoPhotometer, Sl.…”

Identification of genes involved in bamboo fiber development

“…a total of 3.0 g internodal tissue of B. balcooa grown under natural condition using a protocol reported by Rai et al (2010). The quality and quantity of the RNA in each preparation were checked spectrophotometrically (NanoPhotometer, Sl.…”