Can CUT&Tag-enriched DNA be analyzed by qPCR, or do I have to do NGS to analyze my data?

If qPCR analysis is desired, we recommend performing CUT&RUN, as CUT&Tag DNA is not compatible with qPCR. The sample incubation step at 58℃ required for tagmented DNA solubilization in the CUT&Tag protocol breaks open the nuclear membrane, resulting in the final CUT&Tag DNA sample being composed of both target-bound tagmented DNA and background genomic DNA. If qPCR is performed with primers against target genes on the final CUT&Tag DNA sample, there is no way to distinguish the CUT&Tag signal from the background signal.

However, qPCR analysis of target genes is possible using a CUT&Tag DNA library. This is because, during the library amplification process, the tagmented DNA fragments are selectively enriched while the background genomic DNA is diluted out. The qPCR analysis on a CUT&Tag DNA library can serve as a QC step before next-generation sequencing (NGS). We recommend using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag DNA library preparation.

More information on this topic can be found on the CUT&Tag Frequently Asked Questions page. 

Last updated: September 12, 2024