Transcriptome Sequencing

Transcriptome sequencing is also known as RNA Sequencing (RNA seq). Using this approach, the present RNA molecules are analyzed at a certain time point within a sample. Thus, the metabolic state of a certain sample at a distinct time is displayed and reflects the biological state of the cells. Transcriptome sequencing enables the identification of alternative genes, spliced transcripts, post-transcriptional modifications, gene fusions, and changes in gene expression over time, over treatment or between different groups. Thus, with transcriptome sequencing, gene function and gene structure can be interpreted. Furthermore, the function of non-protein coding RNAs can be analyzed. Transcriptome sequencing can reveal molecular mechanisms of disease occurrence and specific biological processes. Additionally, rare or unknown transcripts can be identified, as well as variable cleavage sites and coding sequence single nucleotide polymorphisms.

RNA seq has advantages over other transcriptomic technologies, such as microarrays. It is highly sensitive, allowing for the detection and quantification of almost all transcripts in the cell. Additionally, RNA seq accurately determines each single nucleotide of every transcript. In contrast to, for example, microarray technologies, RNA seq has no difficulties with cross-reactions or background noise from fluorescence signals.

Within RNA seq, different approaches can be used. It is possible to either analyze the whole transcriptome or the coding transcriptome. With whole transcriptome sequencing, coding RNA as well as many types of non-coding RNAs are covered. In contrast, coding transcriptome sequencing targets the mRNA molecules. Its main objective is the quantification of gene expression and the differential gene expression analysis.

Thus, depending on the experimental setup or research objective, total RNA or messenger RNA can be sequenced. Total RNA comprises both – coding and non-coding RNA. We call this product WTS, for Whole Transcriptome Sequencing. The RNA isolation can be done from cells, blood, or even from FFPE embedded tissue. It can be done either by you or by CeGaT. Total RNA contains many different types of RNA, such as messenger RNA, ribosomal RNA, and non-coding RNA.

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.