2.1 Principle of RNA-Seq technology
RNA-Seq is a transcriptome profiling technology that utilizes next-generation sequencing (NGS) platforms.21 The principle of RNA-Seq involve the reverse-transcription of RNA-Seq transcripts into cDNA, ligation of adapters to each end of the cDNA, sequencing and then aligning them to a reference genome or assembling to obtain de novo  transcripts, proving a genome-wide expression profile.22 The quality and quantity of the starting RNA material are the most important aspects to consider when deciding on the methods to generate RNA-Seq libraries.23,24 While working with RNA, it is critical to avoid RNase contamination by using sterile, RNase-free solutions, and plastic ware. It is also important to include quality control (QC) in all stages of operation.23 The method of RNA isolation affects the ability to detect differentially expressed transcripts.25 Various methods of RNA extraction exist in the literature; the most popular and widely used methods are the TRIzol and RNAeasy.25 Other methods include density gradient centrifugation, magnetic bead technology, lithium chloride and urea isolation, Oligo(dt)-cellulose column chromatography, and non-column poly (A)+ purification/isolation.26 TRIzol employs the guanidinium-acid-phenol extraction and regarded as the ‘gold standard’ but chemicals used are corrosive and toxic, whilst, the RNAeasy (and variants) employs the glass fibre filter and silica technology that is fast, simple and safe to use but expensive.24,25
The extracted RNA is treated in solution or on‐column with DNase to remove traces of genomic DNA and to prevent contamination of RNA-Seq libraries by DNA. DNA‐free RNA could then be assessed for both quality and quantity.24 In assessing the quality of the RNA, most laboratories utilize the electrophoretic‐based system.22,24 The quality of the RNA produced is measured through the RNA integrity score, RNA Integrity Numbers (RIN) that varies from one instrument to the other.23,24 The measure reads per kilobase of exon model per million reads (RPKM) and its variance are the most frequently adopted in the measurement of RNA-Seq expression value.27 The acquisition of RNA-Seq data consists of several steps and in each of these steps, specific QC checks are applied to monitor the quality of the data output produced.15