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 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.

  

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.

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