I need to compare gene expression of a gene in two differently treated samples. I isolated total mRNA using a QIAGEN column kit.
1. Do I use nanodrop to measure isolated RNA concentration and do reverse transcription on equal concentrations of RNA or equal volumes?
2. Following cDNA synthesis do I use nanodrop to load equal concentrations or equal volume of cDNA to use as template for PCR to identify differences in my gene expression?
3. Same questions as above for my housekeeping gene of GAPDH.
I assume you want to do a real-time qRT-PCR, using GAPDH as loading control (reference gene). Then the points 1-3 don't matter. This is why one normalizes the mesaurements to a loading control.
More important are following things:
- is GAPDH expression really constant under the experimental conditions you want to compare? Maybe you should consider using several different genes as loading control.
- is the amplification specific and runs with optimal efficiency (for all genes you measure)?
- determine the dynamic range of your assays, so that you can judge if the ct values you measure are trustworthy.
I agree with Jochen, you dont need to worry about points 1 & 2 (there is no reason to normalise the RNA or cDNA), as long as you are normalizing the end data to reference/housekeeping genes (always use 3 reference genes as a minimum, following the MIQE guidelines).
Immediately after extracting your RNA make aliquots so that you decrease the number of freeze/thaw occasion.
I then analyse a subset of my samples using an Agilent Bioanalyzer (ideally the RIN of the samples should be over 5). And then make cDNA, which I dilute 1:2 in sterile water (this can help with RT-qPCR results).
Article The MIQE Guidelines: minimum Information for Publication of ...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line