Eminence RNA isolation:
Total RNA was isolated and quantified by using Nanodrop 2000. DNA was denatured by DNAs, and segregation of eminence RNA from Malnad Rasbale plantlets was challenging due to its abundance in polysaccharides, polyphenols, and many secondary metabolites present in plant samples (Shankar K. et al., 2011). Several commercially available RNA isolation protocols were unsuccessful because of the release of the high amount of polyphenols during the cell disruption process. At first Jaakola et al., 2001 protocol was followed, and also CTAB (hexadecyltrimethylammonium bromide) for RNA extraction for the preceding experiment, the RIN value crossed > 7. β-mercaptoethanol as a potent reducing agent and contaminant absorbents PVP were used to resolve the glitches to get high-quality samples as followed by Wang et al., 2005 and Liao et al., 2004.
CTAB protocol for segregation of high-quality RNA was adapted. For the prevention of polyphenolic compounds and polysaccharides here high amount of β-mercaptoethanol (4%) and PVP (4%) were used in the CTAB extraction buffer. For RNA binding sites, spermidine which is a polyamine that helps in binding nucleic acid was added to compete, and also to compete with all other polyphenol molecules present in samples (Leipold 1977). For the removal of DNA here acid phenol (pH ~ 4.3) in phenol-chloroform extraction and LiCl precipitation were used. The use of 70% ethanol wash for samples played a vital role to achieve RNA purity. RNA extractions methods are mentioned in Table 1. Values of total RNA ranging from 2.0-2.20 and 2.0-2.50 at 260/280 nm and 260/230 nm respectively indicated the efficient quality of RNA extracted. Table 2 indicates OD at A260/280 nm and A260/230 nm ratios for the samples.
RNA integrity determination
The RNA integrity of Malnad Rasbale was investigated with the help of the 2100 Bioanalyzer device (Agilent Technologies). Agilent RNA 6000 Pico Assay Kit (Agilent Technologies) was used. RNA bioanalyzer profile gained from the above-mentioned method resulted in absolute RNA integrity through Ribosomal Integrity Number (RIN) values (Table 3). For the application of RNA for further investigations the obtained RIN values were 5 > which is the good quality of RNA as stated by Schroeder et al. 2006. Visualizing through electrophoretograms helped us to know samples showing high peaks, the size distribution of RNA fragments, gel-like images of RNA fragments, RNA concentration, RIN values, and rRNA ratio (r28S/18S). Through Fig. 1 we can notify the DNA bands of Control, Foc infected, and resistant variety Malnad Rasbale samples. This showed that DNA is almost all free of contamination. Through the recovery of total RNA from fresh weight samples, RNA concentration ranging from 15 to 34 ng/µ was obtained (Control-15, Resistant − 32, and Foc infected-34) as mentioned in Fig. 2A-2C respectively.
Transcriptome analysis using NGS requires high-quality RNA as the main material. To examine whether the RNA output from our methodology adapted is suitable for the technology, we constructed three libraries (Control, Resistant and infected) using TruSeq™ RNA Sample Preparation Kits v2 (Illumina®). Optimization of the kit is for 0.1–1 µg of total RNA, here obtained concentration of RNA was more than enough for library preparation. The quality of libraries was assessed by DNA 1000 kit in a 2100 Bioanalyzer (Agilent Technologies).
cDNA library Construction for NGS analysis:
The cDNA curves of Malnad Rasbale leaf samples generated on 2100 bioanalyzer using DNA 1000 chip (Agilent Technologies) were expressed between 310 and 370 bp. Figure 3A,3B, and 3C indicate the electrogram of the library varied from 4.57 to 34.3 ng/ µl. Peak molarity ranged between 19.5–166 nmol/l and the concentration of samples was from 318–366 bp. Graphical representation indicates curves and length of sequencing libraries constructed with Illumina TruSeqTM RNA sample preparation kit according to the manufacturer’s protocol. The length of DNA fragments was between 310 to 370 bp. Curves were generated on a 2100 bioanalyzer using DNA 1000 chip (Agilent Technologies). Readings are mentioned in Table 4. Using the NGS platform the libraries were sequenced and high-quality de novo sequence assembly was achieved as mentioned in Table 5.
NGS is a type of sequencing technology awfully sensitive to contamination and requires high-quality RNA (Nadiya et al., 2015).To get precise results in gene expression studies high-quality RNA is in need (Yockteng et al., 2013). Using different strategies many commercial kits are accessible for plant RNA isolation, but here more time is required to evaluate samples, and the method works, and the result will be depending on the organism’s tissues studied (Sah et al., 2014). Presently there is no standardized protocol to isolate RNA from Musa Paradisiaca cv. Malnad Rasbale and loomed techniques and methods are according to species and tissue-specific. Consequently, the present study aimed to develop an efficient protocol to isolate RNA from Malnad Rasbale with good quality, high integrity, and purity with high yield by altering some techniques by referring to Jaakola et al., 2001 methods.β–mercaptoethanol and PVP with double concentration as extraction buffer was used. PVP stops browning through polyphenols and also removes secondary metabolites. β–mercaptoethanol denature RNases through reduction of disulfide bonds. This resulted in RNA being good in quality accomplished RNA-Seq library construction.