Hi,
I am currently treating T cells with CD137-targeting aptamer to examine if the aptamers can specifically bind to CD137 receptor to be internalized.
I performed my experiment by treating my T cells (both wildtype and CD137 knockout T cells) directly with the aptamer for 24h. The cells were then lysed, followed by RNA isolation and RT-qPCR to detect the presence of the CD137-specific aptamer. However, CD137-knockout T cells which supposed to be negative) and wildtype T cells show positivity for the presence of aptamers.
I assume that this could be due to the unspecific binding of the aptamers to the cell surface. So I would like to ask for suggestions if there is any RNase product that I can use to remove cell-bound aptamers prior to RNA isolation.
Thank you.
Hi Kang Yi Lee . If it is nonspecific binding (ionic interactions), you could try competing it off the cells with DNA fragments.
Steingrimur Stefansson Hi, so it means that I treat my cells with aptamers together with DNA fragments?
It is possible for me to try to remove surface bound aptamers with proteinase K or RNase prior to RNA isolation?
Hi Kang Yi Lee . I was thinking more about including DNA fragments in the cell wash buffer.
But now that you mention it, having a trypsin or proteinase K step to remove cell surface proteins sounds good as a way to limit non-specific aptamer binding. It is also way more cost effective than to use RNAse.
Steingrimur Stefansson Thank you so much. I will try with proteinase K first and check on other methods if proteinase K does not work well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA