There is some debate about whether RNA concentrations should be equalized before converting it into cDNA. Some scientists say this step improves the consistency of gene expression analysis using qPCR. Others argue that it may change the natural levels of mRNA, leading to inaccurate results.
I believe that equalizing RNA concentrations could introduce bias by altering the relative amounts of mRNA compared to total RNA. This may not be ideal, especially when studying gene expression differences.
What are the best practices for this step? How can we ensure accurate and reliable results?
Hello Ahmed N Al-Saqabi,
No. Instead of normalizing RNA, you should use the same amount of RNA for each cDNA synthesis reaction to ensure consistent starting conditions and avoid variations in reverse transcription efficiency.
Normalizing RNA concentrations before reverse transcription can introduce inaccuracies in the final cDNA quantification.
Since you will be using qPCR, the amount of cDNA is not directly proportional to the amount of RNA used in the reverse transcription reaction. So normalizing RNA before reverse transcription is not necessary.
If you are going to study gene expression differences, use reference genes (like GAPDH) to normalize cDNA levels after reverse transcription rather than trying to normalize the RNA input.
Ensure that your RNA is of high quality and that there is no DNA contamination.
Best,
No, but use the same amount/concentration (i.e. ug/sample) of RNA for each sample.
You can normalize by RNA input or by input volume. If you are using volume then you need to account for that in your methods. Also, spiking in an exogenous mRNA, I've used an IVT RNA in trizol, can track sample loss through extraction and cDNA synthesis. Either way, you need to keep detailed notes on why you chose that method and to have multiple housekeeping genes that are unaffected by treatment.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction