Hi , i am going to try performing qPCR to study receptor expression in mouse pancreas, is their any tips to get a good results? Starting from RNA ISOLATION
Oh, there is a LOT of work before you can do any bench work. qPCR is hard to get correct because folks don't plan it out correctly.
Step 1. Read the MIQE guidelines
Step 2. Determine what type of experiment you are doing - relative expression or exact copy number?
Step 3. Talk with your advisor about the study design. How many biological replicates? How many technical replicates? Which housekeeping gene(s) will you use?
Step 4. Determine exactly how you will analyze your data BEFORE you pipet anything.
Step 5. Determine primer efficiencies with a standard curve.
Step 6. Run a small number of samples with a full set of controls to make sure all controls look as expected.
Step 7. Now you can set up your qPCR with all of your samples & analyze the data.
Expect this to take several weeks to months (depending on Step 5 and or 6).
Extract good RNA with any of the commercial kits.
Integrity of your RNA is the key to success. Check your oligoes and probe in situ. Check your internal control (housekeeping gene which would help you in monitoring your processes. You will also need to create a positive control which would help in quantifying your experimental results.
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