TotalRNAseq
Ribosomal RNA (rRNA) represents >80% of cellular RNA. This “TotalRNAseq” method takes purified DNase treated total RNA as input and removes the rRNA fraction. The MBCF has experience with a number of different kits and protocols, but we have adopted the Roche Kapa RiboErase reagents and protocol as our preferred method. This method uses DNA probes + RNaseH digestion to efficiently remove rRNA and has been shown to perform as well or better than many alternative commercially available solutions.
Pricing
QC + library prep + PE50 sequencing
(50M 50bp read pairs)
DFCI/BWH: $400
External: $500
QC + library prep + PE100 sequencing
(50M 100bp read pairs)
DFCI/BWH: $400
External: $500
RNAseq analysis (VIPER)
DFCI/BWH: +$20/sample
External: +$25/sample
Turnaround
3-4 weeks
Get started
RNA QC
rRNA depletion is approach for both high quality and low quality RNA samples, however the DV200 must be >30% for maximum likelihood of generating useful data.
Analytical Goals
Gene expression profiling, differential expression testing between conditions (replicates required!), pathway, gene ontology and gene set enrichment analysis.
A suitable method for degraded RNA (e.g. FFPE), detecting non-polyA non-coding RNAs, and some splicing analysis applications.
Not suitable for miRNA detection.
Sample Requirements
RNA Amount: 500ng (more is fine, less can be OK too)
RNA Quality:DV200 > 30%
Common Extraction Methods: Qiagen (i.e. RNeasy, miRNeasy) or Trizol. DNase treatment mandatory.
Minimum Input: 50ng
MBCF Optimal Input Amount: 100-200ng (for Kappa)
Volume: >10ul
Concentration Range: 5-500 ng/ul (it will depend)
