Gene Expression Analysis

Overview

Gene expression analysis enables valuable insights beyond the static information of a genome sequence by allowing researchers to detect and quantify the expression levels of multiple coding transcripts between samples using RNA sequencing (RNA-Seq). An understanding of differential gene expression between biological or experimental conditions opens the door to identifying the molecular basis of phenotypic differences. 

Approaches and Techniques

Gene expression profiling typically involves the isolation or capture of transcribed RNA within a sample, followed by cDNA synthesis and library construction and amplification prior to detection and quantitation by RNA-Seq. Alternatively, total RNA input is converted into a cDNA library, followed by hybridization capture of targets of interest. 

mRNA Capture

The KAPA mRNA HyperPrep Kit provides a streamlined, workflow for the preparation of RNA-Seq libraries containing coding transcripts only. Using oligo-dT beads, polyadenylated transcripts are selectively captured from total RNA samples, after which cDNA is synthesized and libraries constructed for Illumina sequencing. Since RNA enrichment prior to library construction relies on the poly(A) tail of mature, eukaryotic mRNAs, this workflow is only compatible with high-quality RNA inputs from eukaryotic organisms. 

RNA Depletion

KAPA RNA HyperPrep Kits with RiboErase (HMR) or RiboErase (HMR) Globin provide for the selective depletion of transcripts (e.g., rRNA, globin mRNA and/or other highly abundant mRNA species), followed by cDNA synthesis and library construction. The enzymatic-depletion chemistry is robust, compatible with both high-quality and degraded inputs (such as FFPE) from all organisms. 

Depletion workflows provide a complete view of the transcriptome, while minimizing the number of wasted sequencing reads from “uninteresting” transcripts.

Targeted Sequencing

Total RNA inputs, from variable quality samples, are converted into cDNA library molecules using KAPA RNA HyperPrep Kits. This is followed by hybridization capture of targets of interest, which requires only a fraction of the read depth of total RNA sequencing. Depending on the design of the capture panel, this approach allows for interrogation of coding content only (e.g., when using an exome panel), or both and non-coding regions. Since it does not rely on intact 3'-poly(A) tails, this method is compatible with RNA of any quality, and from all organisms.