# Input FAQs for the Illumina TruSight RNA Pan Cancer library preparation kit

**The protocol is optimized for 10-100 ng of human total RNA. Can a lower input amount be used?**

Lower amounts may result in low yield and reduced sensitivity, Illumina does not recommend nor support input amounts outside of the recommended range.

**Which method of quality assessment of RNA samples is recommended before starting the TruSight RNA Pan-Cancer library prep protocol?**

Check the quality of the RNA using an Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity RNA Analysis Kit. For more information, see the "[Evaluating RNA Quality from FFPE Samples](https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf) tech note.

**How to choose the input amount to use (in the 10-100 ng range)?**

* If starting with high quality/non-degraded RNA:
  * Illumina recommends 10 ng of high-quality RNA, such as universal human reference.
* If starting with FFPE RNA:

  * The sample input amount is based on sample quality.
  * Use the percentage of RNA fragments >200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality.
  * The protocol has been tested using 20-100 ng of RNA extracted from FFPE as input.
  * The input recommendations for FFPE RNA (20- 00 ng) are provided as a guide for sample-specific optimization.

  ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-23b69c9e488f5a4d772b5ee37c969529951cf856%2Fimage1.png?alt=media)

**Can libraries be made from RNA with a DV200 <30%?**

Success is not guaranteed with poor quality input.

**Are there specific recommendations for input RNA extraction?**

For successful library prep, use an RNA isolation method that includes a reverse-crosslinking step and DNase1 treatment, such as the QIAGEN RNeasy FFPE klit or QIAGEN AllPrep DNA/RNA FFPE kit.

**Which sample types have this assay been tested with?**

This assay has been tested with RNA from blood, bone marrow, and FFPE tissues.

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