In this blog post, we look at RNA sequencing (RNA-seq), why it is so useful, the expanding range of applications, the workflow including critical steps, and associated products.
RNA-seq and its wide-ranging applications
While DNA sequencing only provides the genetic sequence of an organism, RNA-seq describes transcriptome data and other operational information related to the original strand/ observed at the RNA level. This significantly enhance the value of RNA sequencing compared to DNA sequencing. The transcriptome is the complete set of transcripts in a cell, and their quantity. Thus, RNA-seq can be used to understand which genes are turned on in a cell, their expression levels and for a specific developmental stage or physiological condition (see Figure 1). Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues, and for understanding development and disease. The key aims of transcriptomics are to:
- Catalogue all species of the transcript, including mRNAs, non-coding RNAs, and small RNAs
- Determine the transcriptional structure of genes in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications
- Quantify the changing expression levels of each transcript during development and under different conditions
By identification of antisense transcripts and non-coding RNAs, RNA-seq can also be employed to detect mutations, fusion and alternative splicing, post-transcriptional modifications such as polyadenylation and 5’ capping etc. In this way RNA-Seq may be applied to detect early high molecular risk mutations used as cancer biomarkers and potential therapeutic targets, monitoring of diseases, and guiding targeted therapy during early treatment decisions (see Figure 1). Furthermore RNA-Seq is used to characterize and determine expression levels of miRNAs (microRNA), siRNA (silencing RNA) and circRNA (Circular RNA) and other small RNAs, which seem to be involved in the regulation of many developmental and biological processes.
Figure 1: Overview of a typical RNA-Seq experimental workflow. RNA are isolated from multiple samples, converted to cDNA libraries, sequenced into a computer-readable format, aligned to a reference, and quantified for downstream analyses such as differential expression and alternative splicing.
Ref: Griffith M, Walker JR, Spies NC, Ainscough BJ, Griffith OL (August 2015). "Informatics for RNA Sequencing: A Web Resource for Analysis on the Cloud". PLOS Computational Biology. 11 (8): e1004393. Bibcode:2015PLSCB..11E4393G. doi:10.1371/journal.pcbi.1004393. PMC 4527835. PMID 26248053.
NGS RNA-Seq library generation
The RNA-seq workflow for NGS comprises RNA extraction, accurate quantitation, reverse transcription to cDNA, and fragmentation (see Figure 2), except the small circRNA, tRNA, siRNA, miRNA, snRNA, tmRNA, etc. that are sequenced directly after ligation of adapters to both ends. All the steps are critical to obtaining successful sequencing data.
Figure 2: Outline of the high-throughput RNA-seq (HTR) library preparation. In short, frozen tissue samples are ground in the lysis buffer and mRNA is isolated from this using oligo dT beads (1). The mRNA is used to make first and second strands of cDNA (2) and this double stranded cDNA molecules are subsequently enzymatically fragmented (3). The ends of these molecules are repaired and an A nucleotide is added (4) to facilitate TA ligation of the barcoded adapters (5). The ligated samples are then enriched by amplification using adapter specific primers (6) and purified for sequencing.
When choosing a suitable library preparation kit, you must decide whether you want to make a directional or non-directional library. Also, the sequencing platform to be used affects the selection of library preparation methods. Other critical facts to consider are:
NGS RNA-Seq library generation:
The NGS RNA-seq library generation workflow comprises RNA extraction, quantitation, reverse transcription, and fragmentation (see Figure 2), except from small circRNA, tRNA, siRNA, miRNA, snRNA, tmRNA etc. that are sequenced directly. All steps are critical to obtain successful sequencing data. When choosing the library preparation kit, you must decide to make a directional or non-directional library as well as the sequencing platform. The different workflow steps in the process are:
RNA isolation for library preparation:
Commercial isolation kits are available for preparing good quality RNA. One example is NEB's Monarch Total RNA Miniprep kit, which efficiently isolate RNAs of all sizes (>20 nt). The kit includes standard removal of genomic DNA in the column.
RNA quality and integrity:
The quality of the isolated RNA is important, as degraded or cleaved RNA may lead to poor yield or complete failure of library generation. RNA quality is evaluated by determining the RIN value (RNA Integrity number), which should be greater than 7.
Integrity and size distribution of isolated RNA can be checked by electrophoresis on a denaturing agarose gel, staining with a safe nucleic acid dye, such as GelRed. The RNA should appear as two sharp bands corresponding to the ribosomal 28S and 18S subunits, with a brightness of 2:1 ratio (28S:18S) for intact total RNA isolated from eukaryotic cells.
Some RNA's must be fragmented to suitable sizes prior to reverse transcription rather than by fragmenting the cDNA. An efficient method for this is offered by Diagenode's Bioruptor device family, which enables reproducible sonication without the risk of cross-contamination.
Removal of ribosomal RNA and other dominant RNA's:
Most cellular RNA is ribosomal RNA (rRNA), not interesting for research in the light of current knowledge. Therefore, this should be removed before the RNA library generation. Two common rRNA removal options are:
- rRNA depletion
- mRNA enrichment using the Poly(A) tail present in eukaryotes
In addition, globin RNA is usually removed from blood samples and chloroplast RNA from plant leaf samples. NEB offers mRNA enrichment kits, rRNA depletion kits (human/ mouse/ rat and bacterial), globin depletion kits, and a customized RNA depletion kit to be designed for abundant RNA sequences, for which no depletion kit exists.
Reverse transcription and second-strand cDNA synthesis:
Complementary DNA (cDNA) is formed from the RNA template by reverse transcriptase. The first strand of cDNA is made double-stranded by DNA polymerase.
End repair, dA-tailing and ligation of adapters:
The double-stranded cDNA library is treated by End-repair and optionally dA-tailing. Next, the necessary adapters are ligated, making the library ready for amplification and sequencing.
Kits for NGS RNA-seq library generation
BioNordika offers NGS RNA-seq library generation kits for diverse upstream sample sources and downstream platforms and purposes.
New England Biolabs
NEB RNA-seq kits are available for directional (strand-specific) or non-directional RNA library preparation, single Cell/Low Input RNA, small RNA and SARS-CoV-2 RNA. The newly innovated series: NEBNext Ultra II RNA-seq kits utilize streamlined workflows parallel to other NEBNext library preparation kits, making it straightforward to switch between the different applications (see Figure 3). In addition, they are easily scalable and already automated on various robotic platforms. Adaptors and primers (NEBNext Oligos) are available separately, for maximum flexibility.
Alithea Genomics provides 3' mRNA-seq library sequencing kits, based on 3' barcoding of mRNA´s, which enables transcriptomic analyses of bulk RNA from thousands of cells or high sample numbers. This technology named Bulk RNA Barcoding and sequencing (BRB-seq) allows the processing of practically any number of RNA samples in one tube. This significantly reduces both technical variation, turnaround times and costs. The kits offered by Alithea Genomics contains all reagents including oligos and enzymes needed to go from purified RNA to sequencing-ready DNA libraries to be used on Illumina sequencing platforms. Three kit workflow variants are available for: RNA samples, blood samples and cell lysate samples. The blood kit includes globin blockers enabling depletion of unwanted globin genes. The cell lysate kit includes cell lysis buffer, allowing direct cell lysis without prior RNA isolation, making it ideal for screening purposes.