Has anyone cloned the pro-caspase-1 gene from primary porcine alveolar macrophages? Or has anyone seen the mRNA expression by real-time PCR? For cloning, I treated the cells with LPS for 2 or 4 hours as the attached paper says or infected cells with virus (IL-1b production confirmed), but did not work for PCR.
I used specific primers for 5' and 3' end of CDS (http://www.ncbi.nlm.nih.gov/nuccore/AB027296.1) or somewhere in the middle to have smaller fragments. Real-time PCR results did not show the expression, either.
I could clone other inflammasome components (NLRP3 and ASC) and pro-IL-1 beta (even without any stimulation), but struggling with pro-caspase-1.
Do you have any idea about what to try?
Article Molecular Cloning of Porcine Interleukin-1 beta Converting E...
Do you get absolutely no product, or just the wrong product? In my experience differentiating between the different caspases can be difficult with RT-qPCR because the difference between them is quite small.
Hi, Jordan.
Thanks for your reply. I didn't have any non-specific product, either.
For others who might have a similar inquiry,
I actually cloned this gene successfully but not with the primers based on the above sequence.
By running BLAST, I found that there was another sequence available, which has some mismatches on 5' and 3' ends compared to the above one.
So I followed this sequence to design primers.
http://www.ncbi.nlm.nih.gov/nuccore/AK231984.1
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