Getting Started with RNA Sequencing (RNA Seq) – The Basics
RNA sequencing or RNA seq is the process of obtaining the sequence information of a sample’s transcriptome, , including mRNA, rRNA, and tRNA, through Next Generation Sequencing (NGS). RNA sequencing provides information about gene expression in a given cell (in the case of single cell RNA sequencing) or sample set of cells (in the case of bulk-level molecular analysis). This information is important in many areas of research where gene expression is important, such as oncology, including instances of tumor heterogeneity, or virology, including RNA viruses such as coronavirus or HIV.
There are several steps involved in RNA Sequencing:
Sample Collection, Transport, Storage
Before beginning any RNA seq experiment, the target sample needs to be carefully extracted, transported, and stored. Often a DNA RNA shield is used to facilitate both sample transport and storage at ambient temperature.
CellCover is a DNA RNA shield with a non-toxic formulation that works for fast “one step” stabilizing of biomolecules in life science research. It is a gentle tool for RNA fixation, maintaining the status of expression in human and animal cells and solid tissues, including tumors and cultured cells (adherent, suspension, spheroids). DNA, RNA and proteins are all protected.
Calling to mind the computer science expression “garbage in, garbage out” the collection, transport, and storage of samples are perhaps the most important of all. The entire downstream process relies on the integrity of the samples – without proper RNA fixation with a DNA RNA shield, all downstream sequencing and analysis can be compromised.
After RNA fixation with a DNA RNA shield such as CellCover, samples can then be processed for RNA isolation through extraction and purification. There are several kits on the market that achieve this in a relatively simple and straightforward way with centrifugation and a combination of company-specific buffers.
The RNA sample can be left as-is, or can undergo selection and/or depletion, filtering it for the specific downstream analysis intended. Selection of particular RNA species (such as mRNA, rRNA, or tRNA) at this stage will vary by experiment, as well as whether coding and/or non-coding regions are to be included in the transcriptome analysis.
cDNA Library Prep
Once RNA has been extracted, in order to use Next Generation Sequencing (NGS) it must be converted into a double stranded cDNA library via reverse transcriptase PCR (RT-PCR). A library of cDNA is created for sequencing. The specifics of this process will depend greatly on which platform is being used for sequencing, as well as the particulars of the design of the experiment.
Using Next Generation Sequencing (NGS), the cDNA library is now sequenced. The availability and affordability of NGS technology has drastically increased over the last several years with continued innovation. Generally this process takes anywhere between a day and a couple of weeks depending on the scale and complexity of the experiment.
RNA / Transcriptome Analysis
Once sequencing has been completed, it is time to move onto the high throughput analysis step. Just as DNA experiments explore the genome of a given organism, RNA experiments work toward transcriptome analysis, which can be considerably more complicated.
Generally, once the RNA sequencing reads have been obtained, they are either aligned to a reference genome or assembled in a de novo assembly. From there, quantity can be determined and downstream tests can be performed such as detecting allele-specific expression, testing for different expression amounts between samples, or identifying expression quantitative trait loci (eQTLs).