We are trying to knockout a gene in previously frozen human T cells using siRNA. The cells are isolated from human blood and separated from PBMC using negative selection immunomagnetic isolation. They are frozen in cryo-suspension in -80. We thaw the T cells and wash to remove the DMSO and then culture them with the siRNA in a mixture of expansion and siRNA delivery media for 48 hours. We then harvested the cells, isolated RNA, and performed a PCR to validate the RNA knockout. But we're finding that the siRNA isn't reducing RNA count as much as it does in fresh cells.

Does anyone have any tips of how we could get a greater knockout?