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 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.

  

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.

For any feedback or questions regarding this article (Illumina Knowledge Article #3256), contact Illumina Technical Support [e-mail].