The amount of total RNA used by each group was different during reverse transcription. Can it be compensated by adjusting the same CT value during PCR?
You include "housekeeping/reference genes" in qPCR because you can't assume the same amount of cDNA in each biological sample.
You can adjust the amount of cDNA TEMPLATE you add. The Ct value is the data you are collection, you can't adjust that.
Yes, it's generally okay to start with different amounts of RNA in reverse transcription (RT). However, the amount of RNA you start with can influence the efficiency and accuracy of the RT reaction.
Starting with more RNA can sometimes lead to better detection of low-abundance transcripts while starting with less RNA might be necessary if you're working with limited sample material or if you're trying to avoid bias in your downstream analysis.
It depends on what your specific objectives are. Varying initial amounts of genetic material may render your samples incomparable. As Kais Khudhair al Hadrawi pointed out, the efficiency of the cDNA PCR conducted downstream is swayed as the initial concentration of genetic material and other posible contaminants varies.
Regarding the analysis, CT values can't effectively be compensated or "normalized" solely for differences in initial sample concentrations. Alternative techniques could be explored to enhance comparability. For instance, as Katie A S Burnette explained, housekeeping genes can help you deduct a baseline level of gene expression per sample. Other types of internal controls can help you deal with variablity, especially when the experimental desing accounts for it.
Providing more context would be helpful, I'm sure you'll get a better answer that way. Good luck with your investigation!
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