dynamic changes in messenger RNA (mRNA) expression levels, analysis of genetic
variants, splice variants, and protein isoforms [6]. Moreover, NGS based RNA sequencing,
referred to RNA-Seq, could also be used for analyzing non-coding RNAs, microRNA
(miRNA), small interfering RNA (siRNA), and other small RNA classes [11].
During the past decade the basic RNA-Seq protocol for analyzing different types and
qualities of samples has continuously been modified and a variety of optimized protocols
have been released [12, 13]. A basic workflow summarizing the major steps of a standard
RNA-Seq analysis is shown in Figure 11.1. As already described in Chap. 3 the first step
involves the isolation and purification of total RNA from a sample as well as the enrichment of target RNA. In this step, poly(A) capture is commonly utilized to selectively isolate
polyadenylated mRNA molecules. Further, depletion of highly abundant ribosomal
(rRNA) and transfer RNAs (tRNA) helps in mRNA enrichment (see Table 1.1). In the
second step, chemical or enzymatic fragmentation of mRNA molecules into appropriate
sizes (e.g., 300–500 bp for Illumina sequencing) followed by complementary DNA
(cDNA) synthesis is performed. Next, adapter ligation to the 3’ and 5’ ends of the cDNA
is done followed by the creation of a cDNA library. The third step is the actual sequencing
step using a modern NGS technology that generates millions of sequencing reads. Reads
are then quality checked, trimmed, and genome or transcriptome mapping is performed.
Finally, a study specific downstream analysis of data is conducted to investigate and
analyze differentially expressed genes and to perform an isoform identification or genome
annotation [14]. In the following sections we will describe the main steps of a standard and
best-practice RNA-Seq analysis.
11.2 RNA Quality
Extraction of high-quality RNA is a key step in any RNA-Seq analysis. Several RNA
extraction and purification methods (RNA purification involves cell lysis, RNAse inhibition, DNA removal, and isolation of RNA) and commercial kits are available that show
considerable variability in quality and yield of RNA [15, 16]. In general, variabilities in
these steps together with other physical factors, such as the nature of the sample, its stability
and organism determine the quality of the isolated RNA [17]. Therefore, the determination
of RNA integrity numbers (RIN) via Agilent Bioanalyzer or TapeStation systems is a
critical first step in obtaining meaningful gene expression data. The RIN score relies on the
amount of 18S and 28S to assess in vitro RNA degradation and thus RNA quality. It is
highly recommended to use only RNA for RNA-Seq library preparation with a RIN score
between 6 and 10, the higher the better. In terms of lower RIN scores ask an experienced
scientist what to do.
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variants, splice variants, and protein isoforms [6]. Moreover, NGS based RNA sequencing,
referred to RNA-Seq, could also be used for analyzing non-coding RNAs, microRNA
(miRNA), small interfering RNA (siRNA), and other small RNA classes [11].
During the past decade the basic RNA-Seq protocol for analyzing different types and
qualities of samples has continuously been modified and a variety of optimized protocols
have been released [12, 13]. A basic workflow summarizing the major steps of a standard
RNA-Seq analysis is shown in Figure 11.1. As already described in Chap. 3 the first step
involves the isolation and purification of total RNA from a sample as well as the enrichment of target RNA. In this step, poly(A) capture is commonly utilized to selectively isolate
polyadenylated mRNA molecules. Further, depletion of highly abundant ribosomal
(rRNA) and transfer RNAs (tRNA) helps in mRNA enrichment (see Table 1.1). In the
second step, chemical or enzymatic fragmentation of mRNA molecules into appropriate
sizes (e.g., 300–500 bp for Illumina sequencing) followed by complementary DNA
(cDNA) synthesis is performed. Next, adapter ligation to the 3’ and 5’ ends of the cDNA
is done followed by the creation of a cDNA library. The third step is the actual sequencing
step using a modern NGS technology that generates millions of sequencing reads. Reads
are then quality checked, trimmed, and genome or transcriptome mapping is performed.
Finally, a study specific downstream analysis of data is conducted to investigate and
analyze differentially expressed genes and to perform an isoform identification or genome
annotation [14]. In the following sections we will describe the main steps of a standard and
best-practice RNA-Seq analysis.
11.2 RNA Quality
Extraction of high-quality RNA is a key step in any RNA-Seq analysis. Several RNA
extraction and purification methods (RNA purification involves cell lysis, RNAse inhibition, DNA removal, and isolation of RNA) and commercial kits are available that show
considerable variability in quality and yield of RNA [15, 16]. In general, variabilities in
these steps together with other physical factors, such as the nature of the sample, its stability
and organism determine the quality of the isolated RNA [17]. Therefore, the determination
of RNA integrity numbers (RIN) via Agilent Bioanalyzer or TapeStation systems is a
critical first step in obtaining meaningful gene expression data. The RIN score relies on the
amount of 18S and 28S to assess in vitro RNA degradation and thus RNA quality. It is
highly recommended to use only RNA for RNA-Seq library preparation with a RIN score
between 6 and 10, the higher the better. In terms of lower RIN scores ask an experienced
scientist what to do.
146
R. Bharti and D. G. Grimm