I am extracting RNA from various mouse tissues, including spleen and pancreas, for qPCR with the Fluidigm BioMark 96.96 Dynamic Array. As many others have observed, RNA integrity is compromised after DNase treatment. I use the Invitrogen DNase I amplification grade kit. As you can see in the attached image, heat alone (65C for 10min as described in the protocol) causes degradation of 1ug RNA from tissues with high RNase activity, and each kit component alone causes degradation to RNA from tissues with low RNase activity (I followed the DNase protocol but substituted water for the other components. I did also heat the samples to 65C for 10min in each case).
I am considering using the RNase inhibitor RNaseOUT (requires 1mM DTT) to protect my samples during DNase treatment.
Does anyone have any suggestions about how to avoid RNA degradation during DNase treatment, especially for RNA from tissues with high RNase activity?
Edit: I homogenize tissues in TRIzol, phase separate with phase lock gel, mix the aqueous layer with isopropanol, and transfer to an RNeasy column.
The following protocol is used by my current lab, hope it will help. the treatment temperature we used is 37 °C.
RNA with high integrity will be used for DNase treatment to remove DNA contamination. Total 50 ul reaction components included 50 ug of RNA, 10 U recombinant DNaseI (TaKaRa, Japan), 20 U RNaseOUT ribonucleae inhibitor (invitrogen, Japan) and DEPC treated deionized distilled water. Mixed gently and centrifuge briefly, and then incubated for 2 h at 37°C.
Hi Nick,
I had ever done RNAseq on multiple mouse tissues. I used Qiagen's RNeasy kit. They have column which helps to remove genomic DNA. I had no issue on RNA quality. You can give it a try. The only thing need to be careful is not loading too much tissue on each column. Good luck.
we to had same problem, better you use DEPC treated distilled water/for gel run too.
hope this will work out
Hi,
1/ I do not know what RNA yield you have, but if you are able to get something like 50 ug or more, I would modify isolation protocol. I would add extra phenol:chloroform:isoamyl extraction after TRIzol phases separation and extra chloroform extraction prior precipitation.
2/ I do not like any column based kits for RNA extraction, maybe it’s good for small amounts of starting material, but if it is not your case, just go for standard precipitation. Especially I do not trust DNase treatment on the column.
3/ Try to do standard DNAse treatment in larger volume like 50-100 ul, then, instead of heating, do next F:C:I extraction with subsequent chloroform extraction, EtOH precipitation in presence of salt and wash pellets twice with 70% EtOH with short-spins to pick up residual alcohol. In my opinion most efficient DNase is from Roche.
4/ Always use any kind of RNase inhibitors like RNasine (Promega), Protector RNase Inhibitor (Roche) or RiboLock (former Fermentas, now LifeTech), etc.
Best regards,
MK.
Thanks for all your suggestions. I have spoken with other Fluidigm users and learned they also avoid DNase treatment because it degrades RNA. It may be that the above suggestions prevent degradation, but because I have 192 samples, additional steps will be expensive and time consuming, and unnecessary for my purposes. If I test any recommendations above in the future, I will post an update.
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