Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.)

Fatemeh Rahmani,Leila Amraee

doi:10.14720/aas.2020.115.1.692

Abstract

Ribonucleic acid (RNA) quality and integrity are crucial for many studies in plant molecular biology. High-quality RNA extraction from plants with high levels of compounds such as polysaccharides, polyphenols, and other secondary metabolites are problematic. RNA extraction from Lemon balm tissues can be difficult due to the presence of polyphenolic and polysaccharide compounds or can be done by expensive protocols. This study shows improvement of a CTAB-based protocol which allows rapid and easy isolation of high-quality RNA from Lemon balm plant. The RNA obtained is suitable for cDNA synthesis and RT-PCR experiments.

Highlights

The extraction of high quality ribonucleic acid (RNA) is an important step for many studies in plant molecular biology, such as northern blotting, microarray hybridization, both targeted real-time PCR (RTqPCR) analysis and generation sequencing (NGS) (Gambino et al, 2008; Guerriero et al, 2016)
Conventional protocols for RNA isolation usually involve the use of detergents, such as cetyl trimethyl ammonium bromide (CTAB) or sodium dodecyl sulphate (SDS), denaturing organic solvents, reducing agents (β-mercaptoethanol and dithiothreitol), or denaturing agents (Gambino et al, 2008)
Total RNA was extracted with TRIzol (Sigma Alderich), RNX- plus (Sina Clone) and (CTAB)-based protocol

Summary

INTRODUCTION

The extraction of high quality ribonucleic acid (RNA) is an important step for many studies in plant molecular biology, such as northern blotting, microarray hybridization, both targeted real-time PCR (RTqPCR) analysis and generation sequencing (NGS) (Gambino et al, 2008; Guerriero et al, 2016). Phenolic compounds form high molecular weight complexes tend to co-precipitate with RNA by binding to nucleic acids and polysaccharides in the presence of alcohols They contaminate the final extract and interfere with subsequent applications (Gambino et al, 2008). Lemon balm is one of the oldest and widely used medicinal plant in the mint family (Lamiaceae), native to Southern Europe, Mediterranean region and northern Iran (Döring et al, 2014; Jalal et al, 2015) It is a perennial herb and a rich source of natural antioxidants (Saraydin et al, 2012). CTAB-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA from polyphenolics and polysaccharide-rich tissues of old lemon balm

PLANT SPECIES USED

Reagents

ESTIMATION OF RNA QUALITY

RT-PCR

CONCLUSION