I am going to measure the expression of 4 GPCRs in tissue sample from mouse pancreas. How can I analyse the result as I have to compare the expression of my target receptor with other receptors in samples from both knock in and knock out mice. Should I use the same concentration of the primers for all genes including the house keeping genes?
thanks
Sidney T. Sithole
Primer concentrations: You don’t necessarily have to use exactly the same concentration for all primer pairs. What matters is that each primer set is optimized to work efficiently and specifically for its target. Start with standard qPCR conditions, then confirm that each primer pair gives a single, specific product with no primer-dimers. You can use tools from NEB to help you design your primers. Normalization: Use one or more stable housekeeping genes to normalize your data. It’s important to verify that your housekeeping gene expression is consistent across your knock-in, knock-out, and control samples. Relative expression analysis: You can compare target receptor expression between samples using the ΔΔCt method (or equivalent), which accounts for variation in RNA input and reverse transcription efficiency. This method allows you to compare relative expression of each GPCR across your different mouse genotypes. Cross-gene comparisons: Be cautious about directly comparing absolute Ct values across different genes, because primer efficiencies and transcript abundance differ. If you want to compare expression levels between the GPCRs themselves (not just between genotypes), you may need to calculate relative expression using standard curves for each gene. Replicates: Always include biological replicates (different mice) and technical replicates to ensure your comparisons are statistically robust.
Hi Aisha Abdelmalik
Comparing the expression of multiple GPCRs can be tricky. Here are a few suggestions that might work:
All the Best!
0 votes
0 thanks
- Badges
- Science method
More Aisha Abdelmalik's questions See All
Similar questions and discussions