Dear Researchers,
I am going to run RNA Sequencing on mouse brain samples. I have collected the samples and then I put them in the RNALATER solution and stored them at -80.
Following this step, I removed the solution and put a lysis buffer with B-mercapto according to the Qiagen kit recommendations. I followed all the recommendations on the kit.
https://www.qiagen.com/fr/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-mini-kit/?clear=true#orderinginformation
Finally, I have analyzed the RNA integrity and I got 5.7 and 4.4
With the nanodrop, I got 30 ng/ul concentration in one sample and 28 in the other.
Does anyone have any technical advice for me, what could be the reason for the low quality of the RNA?
Attached the report for the RNA migration.
Thanks a lot.
In which volume did you elute the column ? Nanodrop gives not always reliable numbers when nucleic acid are at low concentration. What was the total weight of your sample to be extracted ? Are those embryonnic brain ?
Thanks, Jean for your answer,
I did two 30 ul twice. so 60 ul in total.
The weight of the sample was 25 mg. And they are adult brains.
Best
Mohammed
Hi, maybe you have to wash very well the sample to remove the RNA later. You can compare the extraction of a fresh sample and one in RNA later to understand if you have technical issues (low yield, problem with elution) or can be RNa later problem.
Valeria
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