Trying to lyse a single cell INCLUDING NUCLEUS, reverse transcribe, and qPCR, all in the same reaction, with a purpose of detecting short cDNA and long gDNA fragments. Looks like lysing in the RT-qPCR reaction mix does not give a good gDNA yield. Nuclei are lysed OK in Proteinase K lysis buffer; should I just add Proteinase K to my RT-qPCR mix? Will it affect the other enzymes? Any detergent options I should consider?
Thanks!
Wouldn't a proteinase in your reaction mixture inhibit polymerase activity?
Try to increase your initial denaturation, it may help breaking the cell wall and other proteins so u will get more gDNA.
Priyanka, do you think 0.5% SDS may adversely affect DNA polymerase? Remember that I cannot dialyze or dilute much after treatment with prot.K; DNA polymerization occurs in the same solution. I can only heat-inactivate prot.K.
we are using SingleCellProtect kit developed at Avidin Ltd. for single cells.
It lyses and stabilzes the RNA from cells, which is compatible with cDNA synthesis, so you do not have to purify RNA from single cell or from small number of cells.
It gives much better results (with single cell QPCR, or digital PCR) than other protocols we used.
http://avidinbiotech.com/wp-content/uploads/2015/01/SingleCellProtect_RNA-DNA.pdf
Is it possible to skip RNA extraction and synthesize cDNA directly? - ResearchGate. Available from: https://www.researchgate.net/post/Is_it_possible_to_skip_RNA_extraction_and_synthesize_cDNA_directly [accessed Jul 25, 2016].
- Badges
- Science topic