RNA Sequencing

The CIMAC Network employs several RNA sequencing techniques for transcriptome analysis in various sample types.

The Dana Farber CIMAC in collaboration with the Broad Institute offers Tru-Seq Strand Specific Large Insert RNA Sequencing. The Tru-Seq platform includes plating, poly-A selection and strand specific cDNA synthesis, library preparation (450-550bp insert size), and sequencing (101bp paired reads); sequence coverage is up to 50 million paired reads or 50 million total reads. External RNA Controls Consortium (ERCC) RNA controls are also available. These controls consist of a set of unlabeled, polyadenylated transcripts, 250 to 2000 nucleotides in length, designed to be added to an RNA analysis experiment after sample isolation. The inclusion of ERCCs allows for control of several sources of variability, including quality of the starting material, level of cellularity, RNA yield, platform employed, and batch-to-batch variability. The expected read fraction for ERCCs is around 0.002.

Input Requirements

For FFPE RNA: FFPE, RNA, tissue, blood, stool, or cell pellets that preferably yield >550ng of RNA (12 ng/ul minimum), and a *DV score above 30%.

*DV Score is automatically calculated using the Caliper GX.

For non-FFPE RNA: RNA with either a RIN score above 6 or RQS score above 5.5.

*RNA, tissue, blood, stool, or cell pellets that preferably yield >350ng of RNA.

The Dana Farber CIMAC and the Broad Institute also offer Transcriptome Capture (TCap) an alternative, non-polyA-based RNA capture method developed by the Broad Institute that is optimal for FFPE samples where RNA concentration and quality may be low. This process includes sample preparation (Illumina TruSeq RNA Access) consisting of strand specific cDNA synthesis and target capture, followed by sequencing (Illumina instruments, 76bp paired reads), and a sample identification quality control check (when Sample Qualification of a matching DNA sample is chosen). The TCap method targets 21,415 genes, representing 98.3% of the RefSeq exome. Sequencing yields around 50 million paired reads or 50 million total reads per sample (depending on the reference sequence supplied).

The MD Anderson CIMAC uses Agilent RNA isolation products for RNAseq. RNA will be prepared using suitable purification system depending on sample source (fresh or FFPE). RNA integrity of FFPE RNA will be assessed using either the Agilent 4200 TapeStation and High Sensitivity RNA ScreenTape or the Agilent 2100 Bioanalyzer and RNA 6000 Pico Chip. Either method will employ the region analysis method to determine the percentage of RNA in the sample that is >200 nt for each sample to be processed. It is necessary to have RNA molecules >200 nt for efficient library construction and this value for each sample will determine appropriate conditions at various steps in the workflow. Each RNA sample will be graded according to the following table:

Grade%-RNA >200 ntRecommended Input Amount
Good FFPE RNA>50%200 ng
Poor FFPE RNA20% to 50%200 ng
Inapplicable FFPE RNA<20%Not recommended for further processing
Intact (non-FFPE) RNA>70%100 ng

RNAseq is available at MD Anderson and Dana Farber CIMAC sites.