I am trying to know the expression of a particular gene when treated with a chemical.For that i am doing rt pcr of treated and no treatment wells of cell line.My question is that if I am observing that after certain concentration say 20uM there are some cell death or changed morphology or both in the well.Is it ok to use the total rna from that well to make cdna or not. I use the same rna concentration of each condition to make cdna of all wells(treated or no treatment).or for doing rt pcr all cells in every well should be in healthy condition.
Hi Varun,
It is recommended to use the optimal dose (Not compromising cell viablity, but sufficient to induce gene expression) of chemical for gene expression assays. If there is cell death and morphology change in a chemical treatment, you will find that most of the internal control/House keeping/Reference gene (GAPDH, bACTIN, Tubulin) expression will be drastically different in control and treatment samples. In this case normalizing qRTPCR expression data will be problematic (Questionable).
So even though you are going to use same amount of RNA from treated and untreated samples for cDNA preparation, the expression data may not be reliable. That is why it is recommended that both cells (Treated and untreated) should be healthy for gene expression studies.
Hi Varun,
It is recommended to use the optimal dose (Not compromising cell viablity, but sufficient to induce gene expression) of chemical for gene expression assays. If there is cell death and morphology change in a chemical treatment, you will find that most of the internal control/House keeping/Reference gene (GAPDH, bACTIN, Tubulin) expression will be drastically different in control and treatment samples. In this case normalizing qRTPCR expression data will be problematic (Questionable).
So even though you are going to use same amount of RNA from treated and untreated samples for cDNA preparation, the expression data may not be reliable. That is why it is recommended that both cells (Treated and untreated) should be healthy for gene expression studies.
Hi Varun,
Biswaranjan Pradhan is complete right, you will get Problems with the reference genes. Maybe at first you should make an apoptosis Assay for example with annexinV/ PI to estimate how much cells are death, so that you get an idea of the toxicity of your chemical. And then make at first a time curve to find the optimal time Point to Analyse your gene. And also the function of your gene is important - in case it is involved in metabolic processes it is disputable if this gene is still expressed if the cell is already undergoing apoptosis.
Dear Varun Bansal
My question to you is why cant you use those cells that change morphology with treatment. According to me you can use those cells and the logic behind that is
your treatment is causing change in cell morphology that means it is hampering some normal pathways may be the pathway that you are looking for and you are not observing that change in control cell line. So that does not mean treatment will change the expression of you reference gene also and if in case it change the expression then you can also use the Ct only otherwise use other reference gene. But my suggestion is you go for it and look into the scenario of your target gene and reference gene.
All the best dear
thanks all for your suggestions.So, If my target gene is showing different expression pattern (with/without treatment) and reference gene like beta actin is showing same intensity band with or without treatment,Does that mean that i am doing it right.
Hi Varun,
Yes, you are right, but it is better to have more than one reference gene, especially if you are observe cell morphology Change - beta actin is maybe not the best reference gene. The best ist to have at least two or three reference genes.
Just as an example. In this work we have also examples for what can happen when choosing the wrong reference genes, or only one.
An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells.
Riedel G, Rüdrich U, Fekete-Drimusz N, Manns MP, Vondran FW, Bock M
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