Optimization and comparison of different methods for RNA isolation for next generation sequencing from Elettaria cardamomum

3^rd International Conference on Genomics & Pharmacogenomics

September 21-23, 2015 San Antonio, USA

Nadiya, F1, N Anjali1, A Gangaprasad2 and K K Sabu1*

1Jawaharlal Nehru Tropical Botanic Garden & Research Institute, Thiruvananthapuram, India 2University of Kerala, Thiruvananthapuram, India

Posters-Accepted Abstracts: J Mol Genet Med

Abstract :

High quality RNA isolation is crucial for obtaining meaningful results during small RNA and transcriptome analysis using next generation sequencing. Due to the presence of several primary as well as secondary metabolites, no standard method of RNA isolation is applicable for all plants. The polysaccharides and polyphenols are prominent in various tissues of cardamom (Elettaria cardamomum Maton) which critically hinder the RNA extraction protocols, hence methods using TRIzol, TRIzol with sodium sulphite, RNeasy Plant Mini Kit, PureLink RNA Kit, CTAB, CTAB with PureLink RNA Kit, RNeasy Plant Mini with CTAB, miRNeasy Kit, miRNeasy with CTAB were attempted for obtaining intact RNA from cardamom leaf, stem, flowers, flower buds and young fruits. Our results shown that polysaccharide and polyphenol hindrances are greater in PureLink RNA Kit and TRIzol methods and the amount of RNA generated from all tissues were unsatisfactory. RNA isolated from modified CTAB method yielded RNA with high quantity and good quality as evidenced from A260/280 and A260/230 ratios but the integrity of total RNA isolated was not reliable. The RNeasy Plant Mini Kit and miRNeasy Kit extracted RNA with good purity and high yield but the secondary metabolites present in cardamom interfered the isolation process by lowering the A260/230 ratio. RNeasy Plant Mini Kit with CTAB and miRNeasy Kit with CTAB methods obtained RNA with good purity and quantity as evidenced from A260/280 and A260/230 ratios and BioAnalyzer RIN (RNA integrity number) values. The total RNA from these two methods was found amenable for next generation sequencing.