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Indiana University School of Medicine
Indiana University School of Medicine
Service Cores

RNA-Sequencing

The Center for Medical Genomics considers a variety of factors to decide which RNA protocol to use: Total RNA-seq vs. mRNA-seq.

Total RNA-seq

Normally, total RNA-Seq (also known as whole-transcriptome sequencing) refers to the sequencing of RNA that has been depleted of ribosomal RNA (rRNA), which represents the majority of RNA molecules, both coding and noncoding. Total RNA, when originally isolated, is composed of multiple RNA species, including rRNA, precursor messenger RNA (pre-mRNA), messenger RNA (mRNA), and several types of noncoding RNA (ncRNA), such as transfer RNA (tRNA), microRNA (miRNA), and long ncRNA (lncRNA; transcripts longer than 200 nucleotides not translated into protein). The removal of rRNA in the total RNA-Seq procedure results in improved sequencing data that enables the characterization of these diverse non-rRNA species.

mRNA-seq

Using a selection method to enrich for polyadenylated (poly(A)) RNA, mRNA-seq targets on the messenger RNA molecules (and some long noncoding RNA that are polyadenylated), which represents only a small percentage of the total RNA molecules. If the research goal is to focus primarily on the coding region, mRNA-Seq is the most efficient and cost-effective procedure, and therefore represents the best choice.

When the RNA quality is high (RNA Integrate Number, RIN>7), although total RNA-seq offers broader profiling of the RNA molecules, the data quality of this procedure is normally much noisier than mRNA-seq. Much higher sequencing depth is required, and a much higher percentage of intergenic and intronic sequencing reads are observed. On the contrary, mRNA-seq offers much “cleaner” data. In addition, mRNA-seq can also measure most of the noncoding RNA species that are polyadenylated.

When the RNA quality is low (RIN<7), regardless of the experimental objective, the center recommends total RNA-seq. This is because mRNA-seq primarily targets on the RNA fragments with polyA, this assay will lead to excessive 3’-biase (most signals will be concentrating on the 3’-end of the gene), and the gene expression quantification will be interfered by the RNA quality.

RNA-Sequencing FAQs

If a user’s total RNA is from whole blood, it contains globin RNA. The center is responsible to deplete globin RNA for the user if the user specified it in the sample submission form (additional charges apply). The globin RNA is depleted with QIAGEN QIAseq FastSelect RNA Removal Kit.

No, due to Illumina library preparation protocol, the library insert is more than 150b in size. Therefore, miRNA is not included in total RNA-seq data. Additional miRNA-seq experiments are required for sequencing the small RNAs in the specimen.

This depends on the experimental objective. Unless specifically requested by the users, all RNA-seq libraries are sequenced using 75 paired-end protocols. For gene expression quantification, the center recommends >20 million reads per sample. Higher sequencing depth is desired for alternative splicing analysis and the experiments intended for identifying allele-specific expression or RNA editing.

In collaboration with the IU School of Medicine Bioinformatics Core, the Center for Medical Genomics provides basic data analysis support with $20 per sample. The support includes sequence alignment, gene expression quantification, and differential expression analysis as requested in the initial service request form. Any additional analysis, including additional differential expression analyses that are not initially requested, can be conducted with a fee-for-service model using the standard Bioinformatics Core charge-back schedule.