Hello everyone, this question has confused me for a while.
I am trying to extract RNA from mice brain for RNA sequencing.
However, I just noticed that the original protocol I followed recommend to use glass pestle A and B, like homogenize the sample first with pestle A and then with pestle B for 15-50 times.
For a long time, I homogenized the mice brain cortex by plastic grinder in 1.5 ml tube with homogenization buffer first and then pipetting add up to 1 ml. After centrifuging, I collect the supernatant for RNA extraction by Qiagen RNeasy Micro Kit. But the RIN value is always around 6.
So, I started to wonder whether I used the wrong tool or method to homogenize the brain tissue?
Because of the budget, I can not afford the glass pestles.
I just consider using the stainless grinder.
That's why I want to know the difference between glass pestles and stainless grinder. Will it be the key for the RNA quality?
Thank you.
I don't have experience in treating brain, but in my opinion, glass or stainless or plastic grinder is not the key factor for the RNA quality. keeping the grinder free of RNase and treating the brain sample as soon as possible after taking it out of -80 are more important. We had used stainless balls to homogenize the tissue, and the stainless balls were treated at 180C for 6h to inactive RNase. And why not try liquid N2?
Hello, Lisa.
I do think you will have even better results with stainless steel beads in microfuge tubes like eppendorf safe lock tubes. Systems like MM400 homogenizer (tissuelyzer from QIAGEN) helps to keep samples under low temperature. This is (in my humble opinion) the most important step to achieve the best results in RNA extraction. Of course, minimum time from sampling to extraction and cDNA synthesis.
I did use RNALater but, please, read instructions because, sometimes, people think they should keep samples fromzem with the solution, which is NOT the case. Ok?
One other detail is glass homogenizers need to be kept free of RNAses and treated with RNAses killers from use to use. Another reason I would indicate the use of beads (no reuse) and mechanical homogenizers to do the work.
I hope I shed some light on your question.
Best of luck.
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