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 Qiagen FastSelect rRNA depletion reagents and protocol as our preferred method. This proprietary method uses rRNA specifc probes to block reverse transcription of rRNA sequences during library prep resulting in a fast and efficient means for rRNA removal.
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)