I’m hoping to get some RNA extraction help for getting high quality RNA (RIN>9) for Illumina Sequencing from mouse brain. I’m currently getting RIN 7-8 and here is my process:
Perfuse mouse with cold 1xPBS, take out brain and dissect hippocampus (~15mg tissue). Add 100ul Trizol and snap freeze in liquid nitrogen then keep on dry ice (this process takes about 8 min from perfusion start to snap freeze). Samples are kept at -70C until extraction.
For extraction I homogenize with dounce homogenizer about 10-12 strokes then let sit on ice for about 5 min to dissociate the tissue and then add 20ul chloroform. I’ll wait 2-3 min on ice then centrifuge at 12000xg for 20 min at 4C. I’ll take the aqueous phase and proceed with a Zymo Total RNA cleanup kit (all done at room temp as per instructed by kit) and elute in 40-50ul nuclease free water. I’ll even prepare aliquots for bioanalyzer runs and further use for cDNA prep while the RNA is still on ice before freezing it at -70C.
What can I possibly do to improve the quality from here? I’ve looked into the RNeasy kit for fatty tissue from Qiagen, but I’m just not sure what would be so different from their protocol from what I’m doing now. Does anyone have any recommendations for protocol adjustments or if they’ve used the fatty tissue-specific rneasy kit with qiagen?
Dear Alex,
If you consistently get RNA with RIN betwen 7 and 8 using the procedure you described, I do not think there is much to improve. RIN~8 is already a very good result. Maybe, lower RIN that you expect is related to the tissue itself, or to the instrument/software you use for quality check?
If I have to say something, I would say to expedise a tissue collection to tissue freeze step and disect brain on a very cold surface, but if these would be a problem, you would not get RIN 7-8, so I do not think it will be better.
Personally, I like Zymo kit more than Qiagen, but you may try Norgen also (again, do not think you need).
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