RNA Sequencing

Yes, RNA-Seq is a well-established and accepted method for quantifying gene expression. To see how RNA-Seq compares against microarrays and quantitative PCR (qPCR), view the pages below:

• RNA-Seq vs. Microarrays
• RNA-Seq vs. qPCR

Transcriptomics broadly refers to the study of RNA related to its expression levels, function, structure, and regulation. RNA-Seq is more specific and refers to the technique to study both the sequence and quantity of RNA.

RNA sequencing depth is the ratio of the total number of bases obtained by sequencing to the size of the genome or the average number of times each base is measured in the genome.

Bulk RNA-Seq is a method that analyzes pooled RNA from cells or tissues.

RNA sequencing strandedness allows researchers to determine which DNA strand (sense or antisense) a transcript came from. Compared to regular RNA sequencing methods, stranded RNA sequencing can find novel transcripts, distinguish transcripts from overlapping genes, find antisense sequences, and annotate genes.

Visit the Illumina Stranded mRNA Prep page for more information.

In mRNA library preparation methods, mRNA is selected via oligodT beads from total RNA, so libraries are prepared only from polyadenylated transcripts from samples. In total RNA workflows, rRNA and select other abundant transcripts are depleted from samples, and the remaining RNAs are prepared into sequencing libraries, including polyadenylated and non-polyadenylated RNAs. In enrichment workflows, libraries are prepared from all RNA samples. These libraries are subsequently enriched using an oligo probe panel. Panels can target full coding exomes, transcripts associated with specific diseases, RNA from pathogens, or custom targets. For more information on these workflows, visit the following pages:

• mRNA Sequencing
• Total RNA Sequencing
• RNA Library Preparation

Extracted RNA must be purified and free of contaminants. Illumina recommends following the guidelines provided in your particular RNA isolation kit and selecting an appropriate protocol for your sample type.

The following provides input ranges of RNA for your selected method:

• 25 ng to 1 ug of total RNA input for Illumina Stranded Total RNA and Illumina Stranded mRNA workflows.
• 10 ng to 100 ng of total RNA input for TruSeq RNA Exome, depending on input quality.
• 10 ng to 200 ng of total RNA input for Illumina RNA Prep with Enrichment, depending on quality.

Illumina Stranded Total RNA, Illumina RNA Prep with Enrichment, and TruSeq RNA Exome are compatible with degraded RNA and FFPE RNA input.

RNA-Seq is compatible with all Illumina sequencers. Depending on the library preparation kit, the application, and the data needs, higher or lower throughput sequencers may be more appropriate.

Explore all Illumina sequencing platforms

Contact your sales representative for more information.

Yes, RNA-Seq library preparation methods can be automated. Visit our Library Prep Automation page for more information.

Yes, Illumina offers quality control solutions depending on your application. See our library quantification and QC reference guide for detailed information.