Total RNA Input
The TruSeq Targeted RNA Expression protocol is optimized for 50 ng of total RNA.
RNA that has DNA contamination results in an underestimation of the amount of RNA used.
Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer for samples with an RNA Integrity Number (RIN) value ≥ 8.
Illumina recommends including a DNase step with the RNA isolation method.
Illumina recommends that you check your total RNA integrity following isolation on an Agilent Technologies 2100 Bioanalyzer using an RNA 6000 Nano Chip.
For samples with and average fragment size ≥ 200 bp, Illumina recommends that the RNA input is optimized for 100 ng, of RNA for degraded or FFPE samples.
Illumina recommends running FFPE samples in duplicates.
Illumina requires using a positive control in each batch of FFPE samples being prepared. Using the control enables Illumina Technical Support to troubleshoot in the event that assistance is needed and it cannot provide assistance unless this control reaction was included in the assay.
Illumina recommends using Agilent Technologies Human UHR total RNA (catalog # 740000) as a positive control sample for the TruSeq Targeted RNA Expression protocol.