I will be carrying out some RNA extractions from primary cells using the Qiagen RNeasy Mini kit. I plan to remove the media, wash with PBS, add RLT containing B-me, scrape the cells and then pipette off the homogenate.
Normally I would then extract the RNA immediately, but I have other time-sensitive procedures to carry out, so would prefer to extract the RNA another day. According to the Qiagen manual, one of the postdocs in my lab, and a post on this forum, it will be fine to store the homogenate in RLT with B-me at -80ºC. However, some pages I found on google and another postdoc in my lab don't think it's a good idea. Please can anyone advise me on whether you've tried freezing homogenate in RLT, and whether the quality/yield was good?
0
Qiagen RNeasy kit: Freeze homogenate in RLT buffer with B-me at -80ºC?
Started by jamessmith01, Mar 24 2010 08:01 AM
3 replies to this topic
#2
pito
-
- Active Members
-
- 1,022 posts
Veteran
51
Excellent
Excellent
Posted 24 March 2010 - 08:18 AM
Someone here does freeze his samples and uses them the day after or even later on.
I have never heard him complain about it, however he does prefer to use them directly.
Anyway: there is no real answer on your question I think. All people can say is: try and see for yourself.
I have never heard him complain about it, however he does prefer to use them directly.
Anyway: there is no real answer on your question I think. All people can say is: try and see for yourself.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
#3
merlav
-
- Active Members
-
- 42 posts
Enthusiast
0
Neutral
Neutral
Posted 24 March 2010 - 11:45 AM
We have do sometimes the extraction several days after adding the buffer w/Bme and there is no significant change between the ones do in the same day and the frozen.
Science without religion is lame, religion without science is blind.
Albert Einstein
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie
Albert Einstein
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie
#4
jamessmith01
-
- Active Members
-
- 32 posts
Enthusiast
0
Neutral
Neutral
Posted 29 March 2010 - 06:14 AM
Thanks for the replies. I did store the RLT at -80ºC and got a decent yield form the extraction.
My total yield from 2 x 50 ul fresh H2O elutions (combined) was around 15 ug. Interestingly, in a third elution I got another 10-20% of the yield from the first two elutions (1-2 ug). Not loads, but my labmates were surprised I got anything at all from a third elution.
My total yield from 2 x 50 ul fresh H2O elutions (combined) was around 15 ug. Interestingly, in a third elution I got another 10-20% of the yield from the first two elutions (1-2 ug). Not loads, but my labmates were surprised I got anything at all from a third elution.
Edited by jamessmith01, 29 March 2010 - 06:14 AM.
