After reverse transcription, the addition of adapters and barcodes, and the PCR amplification step, the final library is sequenced on one of our state-of-the-art sequencing instruments. Even challenging samples, such as fragmented RNA, perform well with our total RNA protocols. Since ribosomal RNA comprises more than 80% of all cellular RNAs, we recommend rRNA depletion. We can analyze the transcriptome of mammals, plants, viruses, and bacteria, and we have established many different total RNA protocols for all kinds of applications. The whole transcriptome analysis enables a comprehensive and genome-wide overview of all transcripts in an organism, and genome-wide expression analysis. You can analyze introns, exons, but also regions in between to shed light into splicing mechanisms.
Sequencing of the coding RNA is the most common transcriptome sequencing approach. We call this product CTS, for Coding Transcriptome Sequencing. The messenger RNA has a coding region, which is flanked by two UTRs, so-called untranslated regions, at the 5’ and 3’ end. It carries a 5’ cap and a poly adenylation, the so-called poly-A tail, at the 3’ end. This poly-A tail enhances the stability and the translation efficiency of the mRNA. Most eukaryotes carry this poly-A tail, such as mammals, insects, plants, fungi, and fish. However, it is not present in the mRNA of bacteria. This poly-A tail is essential for the mRNA library preparation. mRNA constitutes only 1-5% of the total RNA. Once the RNA has been isolated from cells or from tissue, the mRNA needs to be purified, using poly-T oligos, which are attached to magnetic beads, and which bind to the poly-A tail. And again, the final sequencing library is then analyzed on one of our modern sequencing instruments.
We can analyze the coding transcriptome of mammals, plants, and other eukaryotes. mRNA sequencing is very sensitive, especially, if you want to analyze mRNA, which is only expressed at very low levels. The main objective of mRNA sequencing is the quantification of gene expression and differential gene expression analysis. It is often used to analyze gene expression levels between different groups, especially when you are planning drug treatment- or disease-related experiments.