6 replies to this topic

[Faun]

Hello everybody!

I would like to prepare some spleen samples for later RNA isolation. In the meantime I was planning to store all homogenates at -20 C. Can I use just PBS (RNAse free) as homogenization buffer (Homogenizer: Mikro-Dismembrator S, Sartorius)and keep RNA intact? After I collect enough samples, I intend to extract viral RNA from supernatant of centifuged homogenates by using QIAamp viral RNA mini kit for isolation of viral RNA from cell-free body fluids (only available in my lab at this time). I will treat that supernatant as it is a body fluid, but I'm wondering if this protocol may work?

Please help me with some advice!

[nightingale]

Hello Faun,

well, if i were you i would homogenize my tissue samples and centrifuge them once received, and then store the supernatant at -80C.

PBS looks a fine choice as a storage buffer, but as a homogenizer am not sure ... sorry for this

see the attached link : Top Ten Ways To Improve Your RNA Analysis Experiment - invitrogen

QIAamp viral RNA mini kit works fine too ...
But,
read the kit insert carefully concerning tissue homogenates, if it includes any specific tips on this ...
for example : homogenizing buffer / storage buffer choice ...
and to check whether to treat your samples as body fluids ( supernatant ) or if there is any mentioning about tissue homogenates specifically.

All The Good Luck.

[bob1]

Store at -80 for RNA.   Snap-freeze the spleens in LN2 (or on dry ice, but it doesn't work so well) first then transfer to the -80.  You can also use a solution called RNAlater if you don't want to extract straight away, but there are maximum dimensions of the tissue that can be put into it so as to allow quick deactivation of RNases.  Spleens are full of RNase so you should have a good protocol sorted out before you start to collect the spleens.

[WSN]

PBS can't be used as lysis buffer, because homogenization releases all the RNases from the tissues, will instantly degrade all your RNAs. Common lysis buffer mostly contain high concentration of guanidine salt, it helps denature most of the RNase, however for long term storage of lysates, you might want to spike in RNASound reagent from Metammune for the prolonged RNA stability in lysates. RNAlater is painful not only in its tissue chunk size limitation, but also in its imcompatiblity with future RNA purification chemicals.

[Faun]

WSN, on 02 December 2010 - 09:53 PM, said:

PBS can't be used as lysis buffer, because homogenization releases all the RNases from the tissues, will instantly degrade all your RNAs. Common lysis buffer mostly contain high concentration of guanidine salt, it helps denature most of the RNase, however for long term storage of lysates, you might want to spike in RNASound reagent from Metammune for the prolonged RNA stability in lysates. RNAlater is painful not only in its tissue chunk size limitation, but also in its imcompatiblity with future RNA purification chemicals.

Thank you for the information!

Can I mix that LyseNow reagent from Metammune with PBS and use it for tissue homogenization, or even better, can I prepare a hommade equivalent? Thank you in advance!

[Faun]

Just to add something....I've found one recipe for GITC buffer, but I wonder if it works well against RNases when used as homogenizing buffer and if it's compatible for later RNA extraction with QIAmp kit?

4M guanidine thiocyanate
25mM sodium citrate (pH7)
0.5% Sarkosyl
dissolved in PBS

[WSN]

The recipe is basically used by every GITC lysis buffer, Qiagen kits most likely are following the same one. The homemade lysis buffer shall be compatible with Qiamp, but just use its lysis buffer. You can drop RNASound into its lysis buffet or simply buy LyseNow RNA kit from metammune.  RNASound won't work in PBS by claims of metammune.com.