Hello!
Does anyone have any experience using a higher amount of fresh-frozen tissue (>20 mg of tissue in RNAlater or >30 mg frozen tissue without stabilizing reagent) for DNA/RNA/miRNA isolation using AllPrep DNA/RNA/miRNA Universal Kit from Qiagen? I used about 20 mg (the maximum amount recommended by the manufacturer) of brain tissue stored at -80 °C in RNAlater, elution was done into 50 ul of EB buffer/RNase free water. Concentrations of both RNA and DNA are quite low (under 100 ng/ul) and also the purity value A260/A230 is quite low at Nanodrop - around 0,6. I guess there could bere a few factors - Nanodrop is not very precise when measuring low conc. samples, also a brain is full of fat so maybe 20 mg is not very much for this type of tissue and also lysing reagent contains guanidium so could this also be the reason for low purity? A260/280 is always around 1,8.
I would like to try more tissue but the protocol says that the column can get stuck because of that.
Thanks for any ideas!
Michaela Ručková Why don't you try using multiple columns (replicates) for the same tissue (i.e. if you have an abundant amount of samples) and then pool the elutes from each column using a minimum volume of the eluent? You may also try to get rid of the excess lipids after the initial homogenization steps to avoid the issues with downstream isolation.
Thank you both for your answers, the concentration is sufficient for further analysis, I was just wondering whether it’s not too low so thank you for telling me that it’s not. Worse thing for me is the low purity A260/A230 around 0,6 … I’m worried whether it could affect further analyses and how could I get higher purity from isolation using this kit.
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