I would like to use 18S RNA as a house keeping gene in real time PCR. Plz share your best experience.
Hi James,
I have not done qPCR on 18S RNA, but I have used housekeeping gene 16S for one of my previous projects related to pathogen detection.
The starting cycle would depends on your template concentration. Higher template would result in lower CT (earlier exponential phase). Having said that, the delta CT (CT gene of interest - CT house keeping gene) should be relatively constant independent of your template concentration (I am assuming your housekeeping gene is part of your template's strands and all samples are under equal treatments).
Hi James,
18S RNA consider to be a very stable HK gene, however, its abundance its a common problem cause you have to be sure that after DNase treatment you have withdraw all the DNA content, other else you will have wrong positive results in HK expression in your samples.
in my opinion, you should search for another HK, cause you have the opportunity to find a easier solution.
Dear Antoni, multiple factors need to be taken under serious consideration, before using the 18s as your sole reference.
This gene is overabundant, as around 80% of total RNA is rRNA. Hence, its ct often appearing in the range of 9 - 12, which is too early. Even the ct above 15 for 18s is usually considered as "poor quality", because its in much abundance that any ct above 15 is logically unacceptable for 18s.
Also to know that any huge difference between your reference ct and target genes ct is also unacceptable. Like, if your reference gene ct is 15 and your target gene ct lands at 28 or 29 per se, so this reference gene (18s or any other) is a poor control for your experiment.
Further, if your reverse transcription cDNA master mix has only oligo DTs primers and does not include random hexamers, so don't try to think of using 18s, as the rRNA, does not carry 3' poly A tail. So oligo DTs wont bind it and your cDNA synthesis eventually amplifies only the mRNA content.
That is why, 18s not represents overall cellular total RNA content, rather it is just the rRNA fraction of a cell.
Since, 18s is highly expressed one, so the cDNA definitely needs higher dilutions usually 1:1000 and above. I have seen in papers that people are using even cDNA at 1:5000 dilution to get a desirable ct range, which can be used as a stable reference.
Also remember, that rRNA transcription has found to be affected by drugs and several biological factors, so, its also not a very stable reference for your qPCR.
Lastly, as a compulsory rule, use always 2 - 3 reference genes to know that treatment, disease or whatsoever your experimental effect are not actually affecting the housekeeping genes you wanna use.
Regards.......
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