High-quality RNA isolation and cDNA library construction from Panama wilt resistant Musa paradisiaca cv. Malnad Rasbale for Next- Generation Sequencing

Abstract

Abstract Protocol standardized for high-quality RNA isolation for gene expression analysis in Foc race 4 resistant banana cv. Malnad Rasbale. Malnad Rasbale was produced by co-cultivating the micropropagated and Foc race 4 infected banana clones with Trichoderma strains isolated from banana farmyards. Existing protocols for RNA extraction from banana cultivars need high-yield and good-quality tissue are perplexing due to manifestation of polysaccharides and polyphenols as major contaminants which hamper RNA isolation. The methodology is based on the CTAB method using a high amount of PVP and β-mercaptoethanol in the extraction buffer. The RNA isolated from resistant banana clones showed high-quality and RIN values in control, Foc race 4 infected and Trichoderma co-cultivated resistant clones were 7.8, 7.6, and 7.6 respectively. cDNA library studies revealed a good calibrated base pair size of 318, 366, and 361 respectively.This investigation is applicable for the molecular aspects of Malnad Rasbale to isolate high-quality RNA, RNA-seq, and cDNA library construction for NGS analysis. CTAB methodology yielded high-quality RNA with RIN value > 7 and good calibrated base pair with > 310 in Malnad Rasbale plants co-cultured with Trichoderma strains in in-vitro developed conditions. This protocol is deciphered for RNA isolation, RNA-Seq, and cDNA library construction.