Illumina Stranded mRNA Prep Ligation Input FAQs

What is the quality requirement of the input RNA?

The protocol is optimized for 25-1000 ng of high quality human total RNA. Other species may require some optimization. Lower input amounts and lesser quality can reduce library yield.

Is the kit compatible with RNA from FFPE samples?

The kit has not been validated with FFPE samples. Using degraded RNA will lead to 3’ sequencing bias, which means only the 3’ end of the transcript will be captured.

What are the optimal 260/280 and 260/230 ratios for the input RNA? (refer to the first informational row, the second and third are provided for context.)

Substance	Absorbance (nm)	260/280 Ratio	260/230 Ratio
Pure RNA	280	~2.0	2.0-2.2
EDTA, Carbohydrates, Phenol	230	<1.5	<2.0
Guanidine HCl	230	<1.5	<2.0

What is the recommended quantitation method for input RNA?

RNA should be quantified with a fluorometric method specific for RNA.

Does input RNA need to be treated with DNase?

Yes, DNA should be removed by including a DNase treatment with the RNA isolation method for sample purity and accurate input quantification.

What is the effect of residual DNA contamination?

DNA can interfere with accurate quantification of the sample. DNA contamination will also impact the strandedness of the sequencing data and increase intergenic reads.

Is there a recommended positive control?

Illumina recommends using Agilent Technologies Universal Human Reference RNA (UHR) (catalog #740000) as a positive control sample.

For any feedback or questions regarding this article (Illumina Knowledge Article #3289), contact Illumina Technical Support [e-mail].