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.
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.
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.
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)
Concentration Range: 5-500 ng/ul (it will depend)