I am running formaldehyde-agarose gel electrophoresis to perform non-radioactive northern blotting. I know northern blotting is a very old story now, however, I am trying to study different transcripts of RNA in my cells under ER and OX stress. Here's my experimental summary:
> I extract my RNA from mammalian cell pellets using Purelink RNA mini kit. I even performed a DNase treatment to make sure there's no DNA contamination.
> I measured the concentration using Nanodrop. The A260/280 ratio is ~1.85 for all the samples. I am loading 20ug of RNA in the gel.
>I've added SYBR safe gel stain to the gel and then ran my samples.
I've ran RNA agarose gels before but the inconsistent RNA loading is something I am seeing recently. Please help!!!
Shruthi Gobbooru See there is nothing wrong to get different concentrations of RNA during RNA extraction because there will be a lot of variability like the amount of cells for each extraction, the experimental factors you are working on, pipetting variability and lots more. I think you are performing a gene expression study by RT-qPCR. For these variabilities in RNA extraction we use reference genes like GAPDH, Beta-actin etc. to normalize the expression levels of the transcripts of interest. Because the expression of these reference genes remains stable.
The easy answer is that you didn't load the same amount of RNA to each well. Double check your math. The 5th sample from the left is so much more concentrated than the rest.
Shruthi Gobbooru See there is nothing wrong to get different concentrations of RNA during RNA extraction because there will be a lot of variability like the amount of cells for each extraction, the experimental factors you are working on, pipetting variability and lots more. I think you are performing a gene expression study by RT-qPCR. For these variabilities in RNA extraction we use reference genes like GAPDH, Beta-actin etc. to normalize the expression levels of the transcripts of interest. Because the expression of these reference genes remains stable.
Let me just say that i think your RNA is beautiful! But for Northern blotting that is required
Going back to your question i agree with Katie
If it isnt a simple error then im not sure. Normally by virtue of contaminants spec readings over estimate rather than underestimate concentration
For that reason i wonder if it is something as simple as Katie suggests?
You could always try independently measuring by fluorimetry to corroborate the nanodrop; but other samples look consistent
Im baffled by this one!
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