I am currently working to generate a certain type of neurons. However not all the cells in the culture will be expressing the specific markers. I would like to check how many cells are expressing the expected markers. Can I used qPCR instead of immunostaining or flow cytometry. Need advice. Thank you.
Dear Teo Ting Ting
For qPCR you need to isolate total RNA, therefore it presents changes in expression of your markers on average in the entire cell population that their RNA were isolated. So this method can’t show how many of cells are expressing a specific maker. In addition, qPCR indicates changes in the RNA level, which may not correspond to the changes in the protein level. Immunostaining shows a semi-quantitative estimation of the fraction of cells that are expressing a particular marker. But flow-cytometry can quantify the population of cells expressing a specific marker.
hi,
-- Flow cytometry will be best to analyze the expression of specific markers in each cell among the whole population.
--Based on the working principle, flow cytometry and qPCR are totally two different techniques. The desired answers can be retrieved from both but in different ways.
-- While qPCR will give the total expression of the markers by the pool of cells. So, qPCR will describe the difference in the expression of different markers based on the step you applied.
--It's like whether you are isolating RNA from the whole culture of cells or at the beginning you have separated the desired cells from the whole culture. If you have separated the different cell types from the whole population, then there you will find particular gene expression prominent while in the whole population the expression may be different.
-- The exact number in terms of cells expressing specific markers is not feasible with qPCR.
-- Similarly, immunostaining will be determining cell pool expression not individual cells.
Regards
Saurabh
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