Laura Melanie BitterlichNational University of Ireland, Maynooth | NUI Maynooth · Department of Biology
Laura Melanie Bitterlich
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Mesenchymal stromal cell (MSC) therapy has seen increased attention as a possible option to treat a number of inflammatory conditions including COVID-19 acute respiratory distress syndrome (ARDS). As rates of obesity and metabolic disease continue to rise worldwide, increasing proportions of patients treated with MSC therapy will be living with obe...
EDIT: The problem turned out to not be with my isolated RNA, my synthesised cDNA, or any of the other components. Instead, I missed that the setting for the reference dye was set to "ROX" instead of "None".
I isolated RNA from cells and transcribed it into cDNA which I then used for a qPCR. In the first run, only a few wells yielded a CT, for most I received an "undefined". I re-ran the experiment with the same result, and I'm not sure what the problem could be, because:
- Both runs resulted in 10 primer-sample combinations for which I had a CT. However, those were not the same combinations both times
- All primers have at least one result with a CT
- All samples have at least one result with a CT
- The primers have worked before
- All are using the same master mix with SYBR Green (with different primers in each mix, of course) which has also worked very recently
- I'm using a template for the run parameters that has worked before
Technically, it should not be the primers, not the samples, and not the master mix. But what else could it be? I consider re-synthesising my cDNA and repeating the experiment, but I'm unsure this will fix anything.