RNA Sequencing- Definition, Principle, Steps, Types, Uses

RNA sequencing is a technique to study the transcriptome of an organism and examine the quantity and sequences of RNA.

As the name suggests, it is sequencing the RNA molecules from the sample using next-generation sequencing.

  • The sector of the transcriptome has been revolutionized by the study of RNA sequencing.
  • Expression of the gene across the transcriptome can be measured as RNA sequencing is a highly accurate and sensitive tool.
  • It provides researchers with adequate data to peek into the RNA sequences related to the undetected changes occurring in a diseased state, under different environmental conditions, and across a broad range of other study designs.
  • RNA sequencing allows the detection of gene fusion, single nucleotide variants, transcript isoforms, and other features.
  • It helps to detect both known and novel features in a single assay. 
  • It has become a powerful tool for analyzing differential gene expression and differential splicing of mRNA.

Principle of RNA sequencing

RNA sequencing is used for studying transcriptomics and gene expression and is a next-generation high throughput RNA sequencing.

From the total mRNA, a cDNA is constructed through the process of reverse transcription, and again it is fragmented. Before sequencing, adaptor ligation and library preparation are practiced. The reading and quantification of the cDNA complementary to mRNA are done by the sequencer.

RNA Sequencing
RNA Sequencing

Steps or procedures of RNA sequencing

  1. Sample homogenization:

The first step of RNA sequencing is breaking down the cells and release of the nucleic acids. For this, different instruments and enzymes can be used like liquid nitrogen, motor, pestle, and buffer for diluting the grounded samples. Then the mixture is centrifuged, and the nucleic content is present in the supernatant.

  1. RNA extraction:

In comparison with the isolation of the DNA, the RNA isolation process is difficult and tedious. The chance of breaking and contamination is high in the context of RNA isolation. So it is very important to be careful and perform the isolation process. For performing RNA sequencing high yield of pure RNA is a must, and its purity is measured using Nanodrop or qubit. Most individuals use silica columns for separating RNA from other components as this column binds nucleic acids. To separate RNA from DNA, they use specific columns that bind only DNA, making RNA free. For isolation of total mRNA from RNA pool, an oligo dT specific column is used. Thus our isolated RNA sample is ready for the next step.

  1. Reverse Transcriptase PCR:

Another step in RNA sequencing needs reverse transcriptase PCR. During this process, the obtained RNA is transcribed into DNA using a type of polymerase known as DNA reverse transcriptase. Finally, cDNA is synthesized from the mRNA.

  1. Second strand cDNA synthesis:

Synthesis of the second cDNA strand is a must after cDNA is synthesized from the mRNA. For the synthesis of the second strand, normal Tag DNA Polymerase is used during conventional PCR. ddNTPs are added to the growing DNA strand by the polymerase enzyme.

  1. Library preparations:

The first step of library preparation starts with the fragmentations, where the whole set of ds cDNA is fragmented by the use of a special restriction endonuclease enzyme. Lastly, during the library preparation adapter sequences are ligated at the ends and amplified for repairing the ends. In the case of mRNA tailing, dA-tailing is optional.

  1. Library purification:

The entire library purification is performed using the DNA purification kit. In the last step, it is essential to purify the entire fragment library, and for the purification, the concentration of the library is accessed using the quantitative PCR or bioanalyzer. Then the sample of fragmented DNA proceeds for sequencing.

  1. DNA Sequencing:

The obtained fragments of the DNA are sequenced using the high throughput NGS machine. This machine reads the sequences as well as quantifies the nucleic acid. Fluorescence chemistry is widely used chemistry behind sequencing the cDNA.

  1. Transcriptome data analysis:

One of the tedious jobs is analyzing transcriptomic data and interpreting the results. The obtained NGS data are huge and more complex as compared with other sequenced data. During this step, the obtained cDNA fragments are sequenced by the use of high-tech machines, and then the data is collected. Next, the flanking regions of the fragments are arranged for obtaining splice variants. After this, the transcriptome data is compared with the reference sequence. Finally, it is analyzed, and the conclusion is driven.

Types of RNA sequencing

Whole transcriptome sequencing

Whole transcriptome analysis detects both coding and non-coding forms of RNA. It is able to measure gene and transcripts abundance accurately. Because of this, both the known and novel features are identified. It helps researchers to identify biomarkers across a wide range of transcripts. To identify the gene entire set of RNA is extracted from the tissue samples. It investigates and explores potential regulatory and transcriptional networks.

Target RNA sequencing

In the target RNA sequencing, the gene-specific, cluster of gene-specific, pathway-specific, or diseases related transcriptomes are mostly sequenced. During target RNA sequencing, less amount of RNA is required and is more accurate than that whole transcriptome sequencing. Also, it is an affordable and highly accurate method for selecting and sequencing specific transcripts of interest.

Enrichment or amplicon-based approaches are used for achieving targeted RNA sequences. In many sample types, detection of both known and novel gene fusion partners, including formalin-fixed paraffin-embedded (FFPE) tissue, is provided by enrichment assay.

Advantages of targeted RNA sequencing

  • Helps in the analysis of specific transcripts.
  • Effective analysis of transcriptome and other pathways.
  • Information on the RNA strand transcripts.
  • Less time-consuming and has the highest data quality.

Small RNA sequencing

In recent years, from animals and plants, many non-coding RNAs have been identified and been seen to play an important role in the regulation of gene expression, cellular roles, RNA processing, and cell proliferation. These non-coding regions are associated with multiple functions in a genome, so it becomes an essential part of studying the small RNAs.

miRNA, siRNA, and piRNA are the small RNA molecule are small RNA molecules that can be quantified and sequenced through the NGS-based RNA sequencing method for various applications. Small RNA is seen in almost every branch of life, which plays an important role in gene expression.

mRNA sequencing

mRNA sequencing is the sequencing of the entire mRNA transcript using the poly(A) tail section which is used for the study of gene expression. Through this sequencing process known as well as novel transcript alterations can be detected. 

It has become of the recent choices for analyzing the transcriptome state of the diseased state, biological processes, and a wide range of study designs. Also, it can identify both known and novel gene fusion and allele-specific expressions.

Advantages of mRNA sequencing

  • It provides more accurate and sensible measurements of changes in gene expression
  • Identifies both known and novel features with a broader dynamic range.
  • mRNA sequencing can be applied across a wide range of species.

Applications of RNA sequencing

The application of RNA sequencing is to determine RNA expression levels. Some of the applications are listed below:

  • Differential gene expression

One of the important applications of RNA sequencing is the comparison of the transcriptomes at different developmental stages, disease conditions, and treatments. For differential gene expression, identification of genes along with their isoforms and expression levels is a must. Some of the tools used for gene expression include Cuffdiff, DESeq, DESeq2, EdgeR, Poisson Seq, Limma voom, and MISO.

  • Variants detection and allele-specific expression

Different variant and allele-specific expressions can be identified by RNA sequencing. Allele-specific expression includes the expression of one allele at a high rate in transcribed mRNA, whereas the other is low transcribed or not transcribed at all. Tools used for variants detection include GATK, ANNOVAR, SNPiR, or SNiplay3.

  • Small RNA profiling

Small RNA is the combination of different RNAs that involves different microRNA (miRNA), small interfering RNA (siRNA), and piwi-interacting RNA (piRNAs). It also includes other types of small RNA, like small nuclear RNA (siRNA) and small nucleolar RNA (snoRNA). Small RNA plays an important role in gene silencing, post-transcriptional gene expression, development, cell proliferation and differentiation, and apoptosis. Techniques used for small RNA profiling include pyrosequencing and other NGS platforms.

  • Characterization of alternative splicing patterns

To understand cell proliferation, development, and human diseases, alternative splicing plays an important role. Alternative splicing pattern is characterized by a powerful tool called RNA sequencing. Similarly, a pair-ended sequence provides information from both ends. The tools used for the characterization of alternative splicing patterns include TopHat, MapSplice, SpliceMap, SplitSeek, GME mapper, SpliceR, GIMMPS, SplicingCompass, MATS, and rMATS.

  • System Biology

Identifying and making the list of differential expression genes is not just the final step. More biological insights can be known by looking at the changes in gene expressions. During this, different pathways and co-expression networks are also identified.

  • Single-cell RNA sequencing

Single-cell RNA sequencing helps to identify intrinsic cellular processes and extrinsic stimuli in the determination of cell fate. It may discover novel species or regulatory processes of biotechnological and medical relevance. Besides this, it is also applicable in stem-cell differentiation, embryogenesis, whole-tissue analysis, disease biology, and treatments.

Advantages of RNA sequencing

  • As this method is not based on probe-based chemistry, so for sequence information designing the probe is not necessary.
  • Gene expression studies can be done more accurately and in a sensitive manner.
  • It has a broader and more dynamic range.
  • It can capture both known and novel alterations of transcripts, even if the sequence information is not provided.
  • RNA sequencing can be applied for any species even if the reference sequence information is not available.

Limitations of RNA sequencing

  • It is a costly and extensive amount of time is utilized for designing, running the assay, and analyzing the data.
  • Protocols for RNA sequencing are not still optimized.
  • Requires high computing facilities like NGS.
  • Splice variants analysis is complex.
  • If paralogues are present, then it becomes more complex to analyze.


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