I have a few questions regarding RNA extraction from cells.
We are trying to extract RNA from cells exposed to two different conditions, so as to understand differences in gene expression.
I know it is important to seeding density for both conditions needs to be the same (say 500,000 cells). Can someone logic out the reasons for this?
Suppose during extraction, one sample loses say 10 to 20 thousand cells, are the results still relevant or should the experiment be repeated?
Thank you
Hi Sathyen,
You want to measure the effect of cell treatment on gene expression. Therefore, you want to avoid other variables that could influence gene expression (like cell density causing changes due to cell division, technical variation during RNA extraction etc.).
Loosing 10K cells during extraction would give you 2% less RNA without other effects.
Herbert
I don't think that 10-20k out of 500k would make a difference that would affect the experiment. Anyway, I would recommend to adjust the culture medium volume and added reagents amount accordingly to have exactly the same conditions.
Hi Sathyen,
You want to measure the effect of cell treatment on gene expression. Therefore, you want to avoid other variables that could influence gene expression (like cell density causing changes due to cell division, technical variation during RNA extraction etc.).
Loosing 10K cells during extraction would give you 2% less RNA without other effects.
Herbert
I don't think the loosing of 10-20 k cells will influence the experiment. The important thing is to quantify the extracted RNA and use the same quantity (for example 1 ug) for retrotransription and then in expression stuydies (Real-time PCR)
I agree with Herbert and Anna, you want to keep conditions between groups as similar as possible to avoid effects due to unknown factors. Also, after RNA extraction you want to quantify RNA and make cDNA starting from the same RNA amount in all your samples.
good luck
Thank you for your answers.
Correct me if I am wrong, but if the gene expression between the two conditions is minutely different, then this variable just might alter the results.
Does this make sense?
Even if I were to convert 1ug of total RNA from both conditions into cDNA and continue with qPCR, would the cDNA produced from the sample with a loss of 20,000 cells be a good representation of the gene expression in that condition?
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