RNA-sequencing (RNA-seq) is a powerful technique used to study the transcriptome—the full range of RNA transcripts present in a cell or tissue at a particular moment in time. Such studies provide valuable insights into cellular behavior, including not only baseline gene expression levels but also differences in gene expression due to environmental conditions, therapeutic intervention, or developmental stage. In addition, RNA-seq can detect mutations, gene fusions, and post-transcriptional modifications.
The first step in the RNA-seq workflow is the creation of RNA sequencing libraries, a multi-step process in which the RNA sample is prepared for analysis on a sequencing instrument. The quality of these libraries is a critical factor in the value of the final sequencing data. This article offers some best practices for handling RNA samples, as well as some tips and tricks for successful preparation of RNA-seq libraries.
Prior to library preparation, RNA is isolated from cells, tissues, or other biological samples. During this process, and during library prep, it is essential to avoid introducing RNases; these enzymes rapidly degrade RNA, compromising the accuracy of results. Before handling the RNA, decontaminate all workspaces and equipment using a solution designed to destroy RNases (available from numerous retailers), and ensure that consumables such as pipette tips, tubes, and reagents are certified as RNase-free. It is also good practice to change gloves regularly.
Maintain the quality and integrity of RNA samples by storing RNA in a -80oC freezer, and by keeping RNA on ice when in use (unless otherwise noted in your workflow). Minimize the number of freeze-thaw cycles (for example, by storing the RNA in small aliquots), and avoid vortexing RNA whenever possible as this can increase fragmentation. Users should also ensure that their input into library preparation is DNase-treated purified RNA and not total nucleic acids. Contaminating DNA into RNA library preparation workflows can lead to downstream issues and less efficient reactions.
Accurate assessment of both the quantity and quality of each RNA sample prior to library prep ensures that the workflow can be optimized to increase the quality of the final libraries. For example, using too little RNA as input can lead to poor-quality libraries that lack biological complexity, skewing the results. Knowing the concentration and quality of each RNA sample enables the optimization of the workflow to increase success; for example, cDNA synthesis, adapter ligation, and PCR amplification steps can be adjusted for optimal library preparation.
RNA concentration is typically measured using fluorometric analysis and/or UV spectroscopy. RNA quality (the extent of RNA fragmentation) is often assessed via electropherogram, which provides a visualization of the size range of the RNA molecules in each sample; electropherogram data can then be used by on-instrument software to calculate the RNA integrity number (RIN) or RNA quality number (RQN). Electropherogram data also enables the user to calculate the DV200, a valuable metric that describes the percentage of RNA fragments >200 nucleotides in length.
Most protocols for RNA library preparation include a fragmentation step to ensure that library inserts are the desired length. Although it may seem counterintuitive when working with degraded samples, it is important not to skip this step; in many protocols, other important reactions are also occurring. For example, the fragmentation buffer included with many kits includes the random hexamer primers essential for first-strand cDNA synthesis; during the initial incubation, the RNA secondary structure relaxes and these primers bind (anneal) to the RNA molecules. For poor-quality samples, the temperature and duration of this incubation can be optimized to avoid over-fragmentation and achieve the desired insert size.
Total RNA samples contain high amounts of ribosomal RNA (rRNA)—sometimes in excess of 95%. However, most researchers are interested in other RNA molecules, such as coding RNA (mRNA) or long noncoding RNAs (lncRNA). Two common methods for enriching for these molecules are mRNA capture, which depends upon the presence of a 3’ polyA tail, and ribodepletion, which employs enzymatic or bead-based approaches to remove rRNA.
When the input RNA is low-quality (with a RIN or RQN below 7.0), mRNA capture can lead to uneven, biased sequencing coverage. For example, mRNAs that have “lost” their 3’ ends will not be captured and thus will not be represented in the final library or sequencing data, and short, abundant 3’ RNA fragments will be captured with high frequency, which can lead to 3’ bias in the results. Thus, ribodepletion is often the preferred method for preparing RNA-seq libraries from degraded samples.
A major challenge associated with processing low-quality RNA is the increased likelihood of adapter-dimer complexes in the final sequencing libraries. These complexes can decrease sequencing efficiency because they bind efficiently to sequencing flowcells and can be preferentially amplified during sequencing. During library prep, the formation of adapter-dimers can be reduced by increasing RNA input or by reducing the amount of adapter used; either of these modifications will increase the number of fragment ends relative to adapter molecules, leading to lower amounts of free adapters. When reducing adapter concentration, the stringency of the post-ligation bead purification can be relaxed by slightly increasing the bead ratio used. This can improve complexity by recovering library molecules that may otherwise be lost.
Roche offers a comprehensive range of products and expert technical support to help you streamline RNA library preparation. More information can be found here.