LCM FFPE protocol version

[Erin Wu]

Hi, I'd like to know why adding proteinase K, then proteinase K inhibitor in LCM FFPE version, is TritonX-100 not enough to lysate tissue?

[Joe Foley]

Yes, we found FFPE tissue very difficult to lyse in a small liquid volume on top of the LCM cap, judging by inspecting the cap on the microscope after lysis, so it only seems to work reliably when we add proteinase K in a long, warm incubation.

On 9/11/20 4:34 AM, Erin Wu wrote:

Hi, I'd like to know why adding proteinase K, then proteinase K inhibitor in LCM FFPE version, is TritonX-100 not enough to lysate tissue? --
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[Erin Wu]

OK，thanks so much for your reply.

I have some questions about the proteinase K inhibitor. The proteinase K inhibitor  stock solutions( EMD Millipore #539470) you used in the protocol  are stable just for up to 1 week at -20°C. It seems not friendly to the long term experiment. So can I use PMSF as a substitute? Will it affact the reaction?

[Joe Foley]

The inhibitor we use is described in doi:10.1016/j.bmcl.2009.01.077 as more effective than previous alternatives, but when I asked Millipore tech support about it (after they'd acquired Calbiochem, before they merged with Sigma-Aldrich) they had no access to any data about the stability of the reagents. You can test it yourself by adding proteinase K and the inhibitor into a regular reaction on isolated RNA. I did this a couple of months ago and found that a solution made from a freshly purchased batch of the inhibitor performed the same as an aliquot that's been frozen in DMSO (but not repeatedly thawed) for four years.

I don't know what would happen with PMSF but it is probably not as effective or stable as the inhibitor that's been working well in this protocol, or it might inhibit other enzymatic reactions in the same tube, and it is also less safe to work with.

Is there a reason you think your inhibitor isn't working? Have you tested it? What problem are you trying to solve?

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[Erin Wu]

I sincerely appreciate your reply. I just searched the inhibitor in the  Sigma-Aldrich website to kok and noticed that they said its solution(resuspended in DMSO) can be stored up to one week at -20℃. So I am not sure if it can promise the long term experiment.

One the other hand, PMSF can be stay longer than that and also inhibit serine protease(eg. Proteinase K). I wonder if it's more cost-effective. But I don't test it. I would like to share my test result afterward. Thank you! 

[Adam Passman]

I've also been concerned about the stability of the inhibitor, but I aliquoted and froze it and I think it's still working fine, but hard to know if it's getting worse slowly.

I was wondering if there'd be an issue with instead doing a heat inactivation of the proteinase K (I think 10 min at 95C?) prior to addtion of the RTase Mix?

[Joe Foley]

A long hot incubation like that would fragment the RNA too much and probably still not inactivate all of the proteinase K.

We are looking into the possibility of using NEB's thermolabile proteinase K (#P8111S) but that requires reducing the temperature of the lysis incubation.

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[Erin Wu]

Joe, have you tried NEB's thermolabile proteinase K (#P8111S)? Does it work equal or more effectively ? I also considered that instead doing a heat inactivation of the proteinase K , but high inactive temperature  may cause RNA degradation. So I also want to try  NEB's thermolabile proteinase K (#P8111S).

[Joe Foley]

I did try it in a tube with pre-isolated RNA and I confirmed that a brief hot denaturation is sufficient to inactivate it so the later enzymatic reactions work normally, without any need for the inhibitor compound and without substantial RNA degradation, but the main problem is you'd have to use a lower temperature in the lysis and I haven't worked through that yet.

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[Erin Wu]

OK, Thanks very much! You mean the main problem is that lower temperature will cause lysis uncompletely or not enough to  reverse crosslink effect from formalin-fixed ? Am I right？

[Joe Foley]

Right.

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