RNA-sequencing (RNA-Seq) has become an increasingly popular technique for transcriptome profiling^[1]. The preparation of high-quality RNA-Seq libraries is critical to the reliability of the sequencing data, in which the integrity and purity of the input RNA play a critical role. Therefore, it is a good practice to perform quality control on the input RNA before proceeding to NGS library preparation. Here, we provide some top recommendations to help preserve and verify the quality of input RNA for more robust RNA-Seq libraries.

RNA isolation is a challenging procedure. Unlike DNA, which is highly stable, RNA is very unstable, quickly digested by RNase enzymes if their activity isn’t immediately inhibited. Traditional RNA isolation protocols are also tedious, time-consuming, and unscalable. Since RNA isolation is critical to many scientific enterprises, researchers have optimized protocols and reagents to speed up and simplify the process.

Anyone working with gene expression, transcriptional regulation or cDNA cloning will undoubtedly have used reverse-transcription PCR (RT-PCR) at some point in their workflow.

RT-PCR is usually carried out as a two-step process: first, total or mRNA-enriched RNA is converted to complementary DNA (cDNA) by a reverse transcriptase (RT) enzyme. The cDNA is then used as a template for PCR.

Welcome back to our RNA-seq article series! Now that we’ve gone through the general workflow and the ins and outs of RNA quality, we will look at some of the popular methods and platforms that you are likely to encounter if you embark on RNA-seq experiments.

Welcome back to our RNA-seq series! In Part 2, we take a look at RNA quality considerations for RNA-seq.

As outlined in Part 1, library preparation is a pivotal part of the RNA-seq workflow. Although the exact protocol will vary according to RNA-seq platform technology (more about this in Part 3), the overall goal is the same: to create a library of complementary DNA fragments that represent the originating RNA sample as accurately as possible and provide information about the RNA features of interest, including information about antisense transcripts, alternative splice variants, miRNAs and other non-coding RNAs, low-abundance transcripts and more.

RNA sequencing or RNA-seq uses next-generation sequencing (NGS) technology to provide a snapshot of the numbers and identities of RNA molecules in any sample at any time under a condition(s) of interest. Since its inception in the early 00’s until now, data gleaned from RNA-seq experiments has revolutionised our understanding of RNA, its roles in animal and plant development, the importance of differential gene expression during health and disease, and how individuals respond to drug treatments. It has also revealed that eukaryotic transcriptomes are much more complex than previously thought!