The kit uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is required. The amount of total RNA needed for QuantSeq depends on the poly(A) RNA content of the sample in question. This protocol was tested extensively with various cell cultures, animal and plant tissues, yeast, fungi, drosophila, and human reference RNA.
As a starting point, we recommend performing the protocol initially with 500 ng total RNA. RNA inputs ≥200 ng are recommended to detect low abundant transcripts efficiently.
The minimum recommended input amount is 1 ng for high quality total RNA (down to 100 pg is possible). Protocol modifications apply for low input (≤ 10 ng) and low quality or FFPE RNA (see FAQ 4 below, and Appendix B and C of the QuantSeq User Guides).
What is the minimum input amount? Are there any recommendations for low RNA input?
When using <10 ng or <1 ng of total RNA input, please follow the recommendations indicated in the table below (also noted in Appendix B of the QuantSeq User Guides):
