How to assess rRNA depletion in the TruSeq Stranded Total RNA workflow

TruSeq Stranded Total RNA (with original Ribo-Zero H/M/R or Gold kits) does not have a quality control post-rRNA depletion. The yield of depleted RNA depends on the amount of input total RNA, rRNA content of the sample, and the method used to purify the Ribo-Zero-treated RNA. Typically, < 8% of the amount of input RNA is recovered (eg, 1 μg total RNA input will yield < 80 ng of RNA depleted of rRNA).

• It is possible to use the the Agilent 2100 Bioanalyzer to quantify the yield of Ribo-Zero-treated RNA. The 5300 Fragment Analyzer System is an alternative.

• Important note: Illumina does not recommend assessing rRNA depletion via traces when using the Illumina Stranded Total RNA with Ribo-Zero Plus, Illumina Rapid Total RNA Prep with Ribo-Zero Plus, or standalone Ribo-Zero Plus workflows.

Method 1: BioAnalyzer trace When assessing the quality of the Ribo-Zero-treated RNA using an Agilent 2100 Bioanalyzer, use the Agilent RNA6000 PicoChip and load 1 μl of the Ribo-Zero-treated RNA. The Agilent RNA NanoChip does not provide sufficient sensitivity. The figure below shows typical Before/After Ribo-Zero rRNA Depletion Traces (Agilent Bioanalyzer Using the RNA6000 Pico Chip)

Method 2: qRT-PCR It is possible to create qRT-PCR primers to the sample species rRNA, along with a housekeeping gene, to evaluate rRNA reduction.

For any feedback or questions regarding this article (Illumina Knowledge Article #2918), contact Illumina Technical Support [e-mail].