Hi all,
Last week, I seeded cells from the same sample into 4 wells (fibroblasts; 300000 cells/well; 1ml/well). well-1 and well-2 are for qRT-PCR test; well-3 and well-4 are for Western blot test.I extracted the mRNA and protein when cells grew into 80% confluency.
However, when I detected the mRNA and protein concentration of these wells, i found that mRNA concentration in well-1 is 65.3ng/ul, while mRNA concentration in well-2 is only 12.9ng/ul. All operation in these two wells are the same. I also found protein concentration in well-3 is 1120.9ug/ul, while protein concentration in well-4 is 496.8ug/ul.All operation in these two wells are the same.
I want to ask are these results normal? or if not, can anyone tell me the possible reasons for this difference. I appreciate any information or suggestion!!!
Aries
I'm not sure how you measured mRNA levels, I will assume you used nanodrop to measure the cDNA to make this inference. With total protein, I will assume you used a total protein assay such as BCA/Bradford.
With that in mind, if you had similar cell seeding density, with real time PCR then the Ct values of your reference gene should come out similar. Based on the 65 & 13 ng values you provided (which again I assume is cDNA), that is around 5-fold difference. Obviously there will be some standard deviation associated, but IMO this is abnormal. If you did real time PCR with a reference gene like Gapdh (or your pick) and saw that the Cts were different then that's a give away that the starting amount of your cells were not the same. Or, the sample with the lowest had extraction/cDNA synthesis issues. Same logic will apply to total protein, the same cell line with same seeding density should roughly give you similar total protein yield. Your total protein differs by roughly a factor of 2, one is tempted to think there is difference in cell density if everything went well for the protein extraction & protein assay.
Hi Can, thank you so much for your suggestions.
In our lab, we use nanodrop to detect RNA concentration directly.
In my experiment, I seeded cells in the same density and I always check this step to make sure nothing is wrong. I also check the cell's growth status in each well, I would collect the RNA and protein samples until cell density is up to 80% confluency.
But I still want to ask, how do you control the cell density difference in your experiment? As I checked cell density using a light microscope, I was wondering whether my operation wasn't right. Or I lost some details when I did that.
Thanks again for all the assistance!@Can Kiessling
We would seed cells by using a cell counter and use the numbers associated with the well type (we used mostly 12 & 24-well plates). Then we used inverted phase-contrast microscopy to confirm the cell confluence by eye estimation (i.e. https://aarmey.github.io/167L-materials/labs/lab6.html). It is a reasonably robust method in the hands of a trained personnel. One thing I noticed with your protein is the high concentration per uL of protein, can you compare your total yield to this one as I don't know your total elution volume - https://research.fredhutch.org/paulovich/en/typical-protein-yields.html
Regarding nanodrop usage for RNA, remember if you are using nanodrop to measure RNA post-isolation (via spin columns, chemical or otherwise) there could be various RNA species included besides mRNA, (tRNA, rRNA, microRNA etc) depending on the method used. If you are using cDNA RNA, then they could be more enriched for mRNA if you used some sort of enrichment method like polyA tailing/oligo dT priming, not sure if random hexamer priming also enriches other RNA species.
Perhaps one of the samples didn't homogenize as well as the other, leading to less extraction? Strange 1-off stuff happens every now and then in the lab, how about a repeat of the experiment with 3 samples and see what you get? Definitely, samples of similar nature should have similar yields.
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