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