> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/rna-enrichment-based-library-prep/library-preparation-rna-enrichment-based-library-prep-faq-list/000003256.md).

# Input FAQs for the Illumina TruSight RNA Pan-Cancer Panel

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

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

**Which method is recommended for assessing RNA samples quality before starting the TruSight RNA Pan-Cancer Panel library preparation protocol?**

Assess RNA quality using the Agilent Bioanalyzer with the RNA 6000 Nano Kit or the Fragment Analyzer with the RNA 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 within 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 RNA.
* If starting with FFPE RNA:

  * The input amount should be determined based on sample quality.
  * Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable indicator of FFPE RNA quality.
  * The protocol has been tested using 20-100 ng of RNA extracted from FFPE samples.
  * The input recommendations for FFPE RNA (20-100 ng) are provided as guidelines and may require optimization for individual samples.

  ![](/files/VjMwU4dVcy9TWE2QtWQL)

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

Successful library preparation is not guaranteed with poor-quality input.

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

For successful library preparation, use an RNA isolation method that includes a reverse-crosslinking step and DNase I treatment, such as the QIAGEN RNeasy FFPE Kit or the QIAGEN AllPrep DNA/RNA FFPE Kit.

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

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

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3256), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003256%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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