For the current experiment I run, I have RNA from three different sets of cells - 1. Untreated 2. treatment1 and 3. treatment2 in which I am looking for the expression of two genes. 18SrRNA is used as the housekeeping gene. Should I look for the expression of 18SrRNA in all the treatments? Or is it enough doing that only in untreated cells?
@Elza Neelima Mathew The internal control gene or housekeeping gene should be used to normalize the gene expression in all the samples (control as well as test), otherwise it would be biased.
One more thing, usually use of 18SRNA as housekeeping gene is not recommended due to its high abundance and therefore it would be better if you could use other suitable internal control gene like UBC, eEF, ACT, TUB.
As Ejaj K Pathan said, you have to measure the expression of the housekeeping gene in all of your samples to do any analysis.
I would recommend you chat with a colleague about experimental design & analysis before you set up any reactions. qPCR is an expensive experiment, if you don't include all of the proper controls, then it's a huge waste of time, money, and precious samples.
Better read up on the MIQE standards for a publishable qPCR experiment.
https://pubmed.ncbi.nlm.nih.gov/19246619/
Hi,
If it is defined as referrnce gene for your organism and treatment, then it should be expressed uniform among your experiment. Use reference gene terminology according ti the MIQE guidelines on your manuscript.
Good luck.
Elza Neelima Mathew I am not sure if rRNA should be taken as a housekeeping gene.
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