Illumina RNA Prep Tagmentation (L) with Enrichment Input FAQs

What is the RNA input requirement?

The protocol is optimized for 10-100 ng of purified total RNA. Lower input amounts can reduce library yield.

Is the kit compatible with RNA from FFPE samples?

Yes, for FFPE samples with DV200 value <80% Illumina recommends increasing the number of PCR cycles to 17.

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 quantification.

What is the effect of residual DNA contamination?

DNA can interfere with accurate quantification of the sample. DNA contamination will also impact the sequencing data, increasing the background level of expression for all transcripts.

For any feedback or questions regarding this article (Illumina Knowledge Article #3293), contact Illumina Technical Support [e-mail].