High RNA quality extracted from the tolerant crop Cyamopsis tetragonoloba (L.) despite possession of low RNA integrity number

Abstract

Salinity is one of the most limiting constraints of crop production. Nevertheless, highly-tolerant crop plants (e.g. ‘Guar’ Cyamopsis tetragonoloba) can thrive well under salinity conditions. RNA-Seq plays a central role in identifying genes involved in the response mechanisms to salt stress in plant species. High-quality RNA, free of genomic DNA is a critical determinant for success in subsequent downstream experiments to shed light on tolerance mechanisms. In this research, an inspection of two different total RNA isolation protocols was employed for preparing highly pure and intact RNAs with good yield from Guar tissue under both high salinity and control conditions. The extracted RNA appeared in all quality metrics to be of adequate quantity and quality for most downstream applications, including high-throughput transcriptome sequencing and expression analysis. However, the outcomes showed that the most widely used RNA quality measurement, the RNA Integrity Number (RIN), is a poor indicator with a weak correlation to the integrity of RNA from this plant. In contrast to human and animal cells, plants have various ribosomal RNA species. Consequently, whenever the RNA was separated on an agarose gel, several RNA bands did appear, which gives the false impression of being unable to analyze the results of the bioanalyzer integrity. Low RIN number RNA samples were further processed by RNA-sequencing RNA-seq experiments from guar. RNA-seq analysis via Illumina’s paired-end method was carried out to trace back and evaluate the extracted total RNA, resulting in high-quality data for subsequent stress tolerance research.

Keywords:

• Cyamopsis tetragonoloba
• Functional genomics
• RNA extraction
• RNA quality
• RNAseq
• salinity tolerance

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Author contribution statement

Conceptualization: AZG, AA, FA; Data curation: AZG, AA; Formal analysis: AZG, FA, AA; Funding acquisition: FA; Investigation: AZG, AA; Methodology: AZG; Supervision: FA, SK, MN; Validation: MT, SA; Writing – original draft: AZG, AA; Writing – review & editing: SK, MN, MT, SA

Additional information

Funding

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number IFKSURG-014.

Funding

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number IFKSURG-014.