Dear all,
I am planning an experiment to perform qRT-PCR, I was thinking about seeding 300,000 cells per well in a 6-well plate, leave it 24 hours to adhere, then add treatments for another 48 hours. The goal is to harvest about 2 million cells, but the question is what about treatments‘ group, will they yield the same amount of cells as the control group? Or, more accurately a good amount of mRNA to be reverse transcriped into cDNA?
N.B: Such treatments and their concentrations are based upon and derived from a cell viability assay made in 96-well plates on 7000 cell per well. And concentrations to be used are IC50, quarter of IC50, and twice IC50.
Many thanks in advance and best regards.
The typical mammalian cell contains 10-30pg of RNA and usually the yield of total RNA from 1 million cells is 10µg, which however, may vary depending on the RNA isolation protocol and the cell type. How much RNA you will finally need depends on the number of genes you intend to analyze by RT-qPCR. If you aim for 1-2µg of RNA per reverse transcription, you may calculate the number of cells you need taking the doubling time and cell death based on IC50 into account.
If you seed 300.000 and aim to harvest 2 million cells you need to know the doubling time of your cells to calculate the final cell yield of the non-treated cells after 72 hours. In the treatment group you will certainly not yield the same number of cells, because otherwise your drug is not working. Hence, as mentioned above, you need to take the cell death rates into account to determine the number of cells to be seeded. You may also consider seeding less or various numbers of cells in the non-treatment group to avoid overgrowth and cell death.
Thank you Dr. Strehl, your answer is of great help. Much much appreciated.
- Badges
- Science topic
- Similar topics
- Clinical Pharmacology
- Dosing