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.

Wondering how to obtain accurate and reliable sequencing data, high-quality NGS library preparation for your research?

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.

Modern genomics seems to be undergoing a shift from using short-read technologies to sequence large numbers of genomes with the aim of detecting SNPs, to sequencing fewer genomes using long-read technologies that can resolve more complex events, e.g., structural rearrangements, copy number variations, and repeat expansions.

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.