# 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.
**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* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003289%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |