I am working on the epigenetic silencing of a gene, upon 5-Aza treatment the transcript level of my gene of interest is going up four-fold compared to the untreated one. Unfortunately, the internal control (GAPDH, 18s rRNA) is also showing an up-regulation (nearly ~ five-fold). What should I do? Is there any other way to investigate the epigenetic silencing of a gene apart from Me-PCR?
How do you know that your gene of interest, as well as Gapdh and 18S are all going up 4 to 5 fold?? What is the reference? It is extremely unlikely that 18S can go up 5-fold, since ribosomal RNA is such a large fraction of the total RNA sample (>50%), it is simply impossible to increase it by that much.
Hi Debasish,
Have you tried to search for a control gene that is unchanged after treatment with 5-Aza? For example, searching GEO datasets (expression database) or GEO profiles might give you an idea on which genes to use as control. You can use a lot of filters to narrow your search. Even using medical subject headings on GEO datasets, for example "Azacitidine"[Mesh] to get very specific records, then combine those with human[organism], etc. A list of available field tags can be found here. http://www.ncbi.nlm.nih.gov/geo/info/qqtutorial.html
This strategy may be less expensive than standardizing another method in your lab.
Bests
Hello Debasish,
I used RPLP0 as a normalization gene in a similar set of exepriemnts and did not obseve any changes in RPLP0 expression. We use a recommmended Taqman assay from Applied Biosystems. Good luck!
How do you know that your gene of interest, as well as Gapdh and 18S are all going up 4 to 5 fold?? What is the reference? It is extremely unlikely that 18S can go up 5-fold, since ribosomal RNA is such a large fraction of the total RNA sample (>50%), it is simply impossible to increase it by that much.
When your internal control goes up or down upon a treatment you can’t say anything. How do you know it goes 4 fold without normalization? Normally the GADPH, 18 S shouldn’t vary that much with 5-aza. So, there is something wrong in your procedure. Maybe the extraction efficiency or the integrity of your RNA preparation is not the same between the two samples. Likely, it is your quantification which is not accurate…
You need an internal control for your RNA expression levels. There was a thread discussing the issues of normalizing RNA expression using house keeping genes due to the variability of their expression as a result of drug treatment or even across different tissues. Some people suggested sparking the RNA preparation with exogenous RNA and I have seen a high impact publication which validated this approach (I can look for it). Ortherwise you can try Maria's control. Remember changes in the pattern of expression of your gene upon 5Aza treatment is only a start.
I agree with David, how it is possible for the 18S to increase four-folds? I am assuming that you used qRT-PCR, correct? Which method you used, SYBR green or TaqMan??
Dear Magdy and David: Thank you for your valuable suggestions.. yes we are using SYBR green and moreover we assume the increase of fold expression by the means of ct value, as the control ct value is 18 and the treated one is 12.. so we are assuming.. i am a nobish in this field and started my gard just 6 months so it will be a great help if you can suggest me an ideal template protocol that i can try..
The simplest explanation is that the concentration of RNA added to the "treated" reaction was 4-5 times more than what was added to the "control" reaction. So you need to re-visit how you measured the concentration of RNA and probably also run a gel on the RNA (a simple Ethidium bromide stained agarose gel will suffice) to make sure that the quality of the RNA in both samples is good. The gel image would also help ensure that your concentration measure is ok.
As David recommended, check the RNA quality and quantity. Make sure to use high quality RNA (with bio-analyzer RIN = 7 and higher ) . Measure the concentration of 2 ul of the sample using a standardized nano-drop, measure it three times and take the average. Also. make sure you primers are very specific and not picking other genes. You can look at the melting curve at the end of the QRT-PCR run. If your lab can invest some money, use TaqMan assay.
@Jian: I have quantified the expression level of the gene, as we know that 5-Aza is a demethylase than if my GOI is methylated in control it have a chance to go up in the 5-Aza treated cell line..
Just looking at expression levels of your gene will say nothing about epigenetic silencing or even methylation. if your goal is to study changes in DNA methylation, you need to look at this directly. 5-Aza could decrease methylation at an activator which effects your target gene, thus indirectly increasing expression without necessarily changing DNA methylation at your target. You might want to rethink this project ad discuss this with your mentor.
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