I've collected some cells using FACS and want to compare different cell populations on microarrays. I sent my cells directly to the microarray service company and had them do the RNA extraction and capillary electrophoresis before going through the pro synthesis and microarray processes.
The capillary electrophoresis results clearly show that the RNA quality is good, which is good news. However the concentrations calculated from the electrophoresis results are very different from the readings of Nanodrop. Specifically the Nanodrop readings are about ten times higher then what the capillary electrophoresis indicted.
The microarray service company is very confident about the Nanodrop results and told me that the sample is ready for low input probe synthesis, no extra amplification is needed.
However I am still concerned. Can someone confirm that at ~50 ng/uL range, Nanodrop still gives reliable readings while the contraction estimated based on capillary electrophoresis is not trust worthy?
If the service company used CE or Agilent Bioanalyzer RNA chip for the integrity and quantitation analysis then I would suggest to go with those results than Nanodrop.
Personally I would take the Nanodrop results with a grain of salt. It is a spectrophotometric technique which isn't exactly the most precise tool for absolute quantitation. I use Nanodrop mostly for ball park estimation on the quantity, as well as rapid method to judge the extracted RNA's 260/230 and 260/280 ratio.
Look at the nanodrop scan especially the 260/230 ratio. 260/230 should be about 2.0. If lower then the NanoDrop estimate could be inflated especially if there is no valley between the 230 and 260 peaks.. We normally see this as a problem if conc.
When the sample is pure, Nanodrop is better, since the nucleic acids are not separated by length, the signal-to-noise ratio is better. For very low concentrations one may use an RNA-specific dye to increase the sensitivity and reduce bias. But then, recording of an appropriate standard (calibration) curve is neccesary.
with good quality RNA, confirm by migration, i almost have the same quantitation result between nanodrop and Bioanalyzer for a 50ng/µL RNA solution
Even Nanodrop is not that trustworthy. You have to check the quality of you RNA sample. I forget the name of the method, but if you want to know, I can find out for you.
Bioanalyzer gives you a "ratio" of the S-RNA (RIN) on a scale from 0-10.
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