I recently worked on Acetylcholine esterase 1 (AchE) gene expression and Cytochrome p450 (CYP6AA6) through Rt-qPCR using designed primer to produce partial gene PCR product in field resistant populations of Anopheles. I found that all tested Anopheles could express AchE, while only few could express CYP6 AA6.. What could be the reason behind this different gene expression?
I had used b-actin as control for the study
Have you tried any different primers? It is possible that some lines might have differences in the primer binding sites. It is also possible (probably more so depending on if the primers were designed to conserved sequences) that the differences are the result of actual changes in gene expression between individuals. Assuming the latter, the question becomes why they have differences. It could be due to differences in environment or differences in genes (either in the regulatory sequences of the p450 gene, or in other proteins (transcription factors, receptors, signal transducers, etc) that control the expression of that p450. You should try growing expressors and non-expressors side by side under controlled conditions to see if the difference is the result of genetics or environment.
If the primers were for conserved regions, check whether they were from the same environment, otherwise grow them under similar controlled environment and see expression profiles. If difference still exists analyse the primer binding sites for alteration of sequences and if no alterations found, analyse the regulatory regions.
Good luck!
a) existe grande probabilidade dos "primers" não estarem anelando no mesmo local Primeiramente eu me certificaria se os "primers" foram devidamente desenhados.
b) certifique-se de seu controle positivo no PCR
c) reveja os dados desses indivíduos que apresentaram padrão diferenciado e verifique se ha alguma informação nos dados que lhe chame atenção.
d) o ambiente (controlado X condições normais) poderia estar sendo um fator de confusão, mas essa seria minha ultima explicação, ou seja, alguma molécula em condições ambientais adversas estar interferindo na expressão do gene.
@David Krishnan and Everaldo: Thank you so much for your help
I have used b-actin as control for the gene expression.
Yes, I have used four primers on cytochrome P450. There were very limited cytochrome CYP6 gene sequences available in NCBI databases in Anopheles.
With the limited resources, I initially designed 2 degenerate primers designed from the conserved amino acid which gave very unstable result for a very very long time, therefore I was compelled to use sequence specific primers.
At present I have 2 sequence specific primers as CYP6AA6 and CYP6F1. CYP6AA6 primers could give product in four species while CYP6F1 could give in a single species. I had run the PCR thrice, and got the same result in all times.. How can I try growing expressors and non-expressors ? I am araid I dont know how..
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