The TruSeq Stranded mRNA Sample Preparation protocol is optimized for 0.1–4 µg of total RNA.
Total RNA Input
Lower amounts of RNA may result in inefficient ligation and low yield.
The protocol has been tested using 0.1–10 μg of high-quality universal human reference total RNA as input.
Use of RNA from other species, tissues, or qualities may require further optimization with regard to the initial input amount.
The protocol recommends diluting the in-line controls for tracking the steps involved in converting dsDNA into libraries.
The dilution is optimized for 0.1–4 μg of high quality input RNA.
When using less RNA or RNA with very low mRNA content, these controls might need further dilution.
If no controls are added, use Resuspension Buffer in place of the controls in the protocol.
It is very important to know the quality of the RNA starting material. The fragmentation conditions were optimized for high quality RNA.
Illumina does not recommend the use of low quality or degraded RNA with this protocol. Use of degraded RNA can result in low yield, over-representation of the 3' ends of the RNA molecules, or failure of the protocol.
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 greater than or equal to 8.
RNA that has DNA contamination will result in an underestimation of the amount of RNA used.
Illumina recommends including a DNase step with the RNA isolation method. However, contaminant DNA will be removed during mRNA purification.
Alternatively, you can run a formaldehyde 1% agarose gel and judge the integrity of RNA upon staining with ethidium bromide.
High quality RNA shows a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb.
Both kb determinations are relative to a RNA 6000 ladder.
The mRNA will appear as a smear from 0.5–12 kb.
Purified mRNA Input
You can also use previously isolated mRNA as starting material. Use the entire fraction of mRNA purified from 0.1–4 µg of total RNA.
Illumina recommends using Agilent Technologies Human UHR total RNA (catalog # 740000) as a positive control sample for this protocol.