When I did the qPCR experiment using the TLR4 assay on spinal cord RNA of TLR4 KO mouse, the TLR4 mRNA was measurable. I did the genotyping of this tissue sample, and it was a homozygous clean KO TLR4 mouse. Any ideas why this might happen?
I few ideas:
Contamination in your qPCR reactions. What did your controls look like?
Amplification of a second, similar yet related gene with your qPCR primers. Is TLR4 a single-copy or part of a gene family?
The "knockout" creates a nonsense mRNA that is transcribed, but not translated.
Your genotyping PCR is not correct (possible, but not likely if you have good controls and knowledge of the mouse breeding).
Good luck!
Hello Alaa.
Have to obtain detailed history of gene construction and whether the original study has used the identical primer pairs. Which part of the TLR4 is deleted and whether the other TLR family gene have no homology to your primer pair sequences.
It is no doubt that specific antibody to TLR4 is the best to tell by Western blot. Any specific antibodies?
I suppose that the KO mice are not tissue-specifically controlled with other means. If it is tissue-specific KO, make sure that your embryonic or neonate or adult tissues isolated are absolutely free of other tissues.
Best wishes.
Thanks a lot for the very previous answers. It seems that there is part of the mRNA synthesised, however, not translated to TLR4 protein.
I am thankful for your help!!
Best regards, Alaa
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models