I have run RNA samples in 1% agarose gel but I cannot visually see a ratio 28S/18S ~ 2. What I might be doing wrong? Also, the samples marked with numbers, should I still consider them for qPCR?
Could be synthesize cDNA from isolated RNA sample . some time 28s and 18s bands are not visualised due to presence of ethanol in isolated sample.
Could you provide a sharper image? It would also be nice to quantitate the bands as 28:18 ratio should be approx. 2:1.
It is hard to make a qualitative statement without seeing the presence of 5S bands.
Based on first impression, I would be skeptical about the integrity of sample 10 for the first image. The band below the 18S is more sharply stained than the 28 & 18S.
http://www.lcsciences.com/discovery/technical-bulletin-customer-quality-control-checks-of-rna-sample/
https://www.thermofisher.com/tr/en/home/references/ambion-tech-support/rna-isolation/tech-notes/assessing-rna-quality.html
I am extracting from mouse white adipose tissue, with DNase I for elimination of gDNA.
Apart from samples 14 and 21, all samples have ratio A260/280 ~ 2 and A260/230 >1.8-2.1, although I don't the quality of RNA will have much influence on its integrity on the gel.
I tried to quantify the bands (ImageStudio SW) but how can I reach precision if when I manually draw the area of selection..a few mm more or less already changes a lot the ratio?
Try to see if you can import the image and analyse using this software:
https://www.licor.com/bio/products/software/image_studio_lite/
After DNase treatment it is bit hard to visualize on gel the ratio (2:1). I suggest you can analyse the suspected samples once again on the gel with equal quantity of RNA based on nanodrop and then decide. Otherwise I think you could reextract only samples 3 and 10. Further you can also check the qubit readings.
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