I would like to confirm PCR Results, so i thought that "knock out" of gene of interest will work. Who support this? If someone has different aspect would you please share?
If the gene is essential you would have to use a conditional knockout.
Hi Kiriakos!
I personally think that the KO is not necessary. I may suggest some alternatives that seem easier and faster than doing a knock-out:
1) As Sebastian Schmitt
mentioned, perform a PCR with primers that anneal to a different region of the cDNA or Western Blot to check the protein levels (herein seeing if the expression is not only at transcription level, but also in protein level).2) Treat cells with ciclohexinimide to stop the transcription and check if the transcripts of your genes are stable are still have a good concentration.
3) Check the expression (via RT-qPCR for example) of another gene pathway-related to the gene you are looking for. Example: if you are checking the levels of a gene that encodes for a transcription factor, check if any of the genes that pressumably regulate your transcr. factor are also expressed.
In addition you can perform functional assays, but it depends of the protein you are checking (if it's a NADH-depending enzyme you can do spectrofotometry, if it's a kinase check the phosphorylation activity, etc.)
What gene are you trying to confirmate its expression??
I hope it helps!
Do not hesitate to contact for further info or diving into this science topics
Francesc Estrany
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