I am trying to check downregulation of a gene using real time pcr. Real time pcr shows that the gene haa been downregulated in my test samples and i get very less or no amplification for the gene in the test samples.The controls are fine. But on the other hand i m getting amplification in conventional pcr, which shows the expression level is same in control and test. I am confused. Please help
There may be an issue with the real-time PCR assay, such as suboptimal primer design, low quality or degraded RNA, or a technical issue during the assay. It's important to verify that the real-time PCR assay is working properly by confirming that the positive control amplifies as expected and the negative control does not amplify.
Another possibility is that there may be differences in the specificity and sensitivity of the real-time PCR and conventional PCR methods. Real-time PCR is generally considered more sensitive and specific than conventional PCR, but it is possible that the real-time PCR assay may be more prone to false negatives or other technical issues that are not present in conventional PCR.
It is also important to consider the experimental design and the biological context of the samples. Are the test samples treated differently than the control samples? Are they from different tissues, cell types, or developmental stages? These factors could affect gene expression and may explain the observed differences in results.
To troubleshoot this issue, I would recommend running additional controls and replicates to confirm the results and rule out technical issues. It may also be helpful to consider alternative methods for validating the downregulation of the gene, such as RNA sequencing or western blotting.
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