Optimization of an Efficient RNA Isolation Protocol from Fusarium wilted Pigeonpea Plant (Cajanus cajan (L.) Millspaugh)

Abstract

Aims: The present study demonstrated a comparative evaluation of RNA isolation from pigeonpea by using conventional or modified laboratory based methods as well as optimizing the existing well known protocol to extract a satisfactory amount of purified and reproducible RNA required for downstream processing. Study Design: Experimental. Place and Duration of Study: Plant Tissue Culture and Biotechnology Laboratory, Department of Botany, University of Kalyani, Kalyani, Nadia, West Bengal, India in 2015. Methodology: In the present investigation, an attempt was made to optimize the method for getting high quality RNA from healthy pigeonpea seedling as well as inoculated with fungal strain of Fusarium udum (FU) Butler. After optimizing the method showing best performance among all experimenting protocols, the RNAs were further validated for quality by reverse transcription polymerase chain reaction (RT-PCR) for amplification of plant specific Ascorbate peroxidase           gene (APX) and FU specific Cellobiohydrolases (CBHs) gene responsible for infectivity of     Fusarium udum. Results: The determinant applications of Guanidium thiocyanate (GITC) and sodium citrate based lysis buffer for efficient extraction at initial step and an effective precipitation by using isopropanol and sodium chloride in the final step made a successful optimization of the excellent quality of total RNA isolation. The same RNA was self sufficient to identify the traces of plant and fungal specific gene through RT-PCR. Conclusion: This observation under certain modifications could be effective to get RNA of excellent quality with the elevated yield from extremely challenging high polysaccharides and polyphenolic rich pigeonpea plant. Simultaneously this experimental design will not only provide better options for the identification of rare transcripts involved in resistance mechanisms in pigeonpea against various stress responses, but also make an opportunity to study the plant pathogen interaction at the molecular level.