Hi,
I have isolated some mouse splenocyte and collected the pellets after isolation. I added QIAZOL into the cell pellet, but I forgot to pipette it up-and-down before I put it into the -80 freezer. The yield of the RNA extraction was not very good after that. Is it still possible to do something at this point or it is all gone? If I transferred the QIAZOL/ Cell pellet into another round bottom tube and use homogenizer/ round beads to destruct the pellet+ QIAZOL, is it still helpful? So sad to notice that I should have homogenized the cell pellet before I froze them.
Thanks for everyone's input.
Dear Ichun,
Try to repeat your RNA extraction with a new pellet. Just try to mix the pellet with trizol next time.
Good luck!
Hi Chen
check this out
https://www.thermofisher.com/in/en/home/references/ambion-tech-support/rna-isolation/general-articles/ten-ways-to-improve-your-rna-isolation.html
You might have thawed your sample..This step will kill your RNA .
Dear Hamadi Madhi,
I will definitely remember to do so next time. However, those cell pellets are from mouse after beads separation.....I am looking for ways to save them at this moment....I might still need to start over again afterwards, but I would try my best to save the samples...
Dear Karthikeyan Rajagopal,
So, do you have other suggestions if I really forgot to homogenize my pellets after I added Trizol and just froze it...?
Thanks for all your recommendations!
I-Chun
Dear Chen,
Just try to extract your RNA from the remaining pellet. Maybe It might work.
Please try to share your result with us!! I am really looking for that.
Good luck.
Best,
Hamadi
trizol like reagents does not really preserve cells and enzymes... i will go further, anyway. check at least some house-keeping target, how is levelled in samples.
Hi Ichun,
Attila is right...trizol does not preserve RNA, but you did preserve it at -80ºC, so I would be confident in saving them. After defrosting pellet+quiazol, homogeneize sample in a teflon homogenizer or something like it....and wait 5min at RT to assure cell lysis! Afterwards proceed to RNA extraction protocol. Maybe it works!
Good luck
Adriana
Thanks for all of your recommendation, but the yield was really bad.
I have tried to thaw the QIAzol/ pellet and started pipetting before it all thawed.-->Not helpful.
I also tried to do the above and transferred them to a round-bottom tube. Then, I used bead+ homogenizer at 30Hz for 2 minutes. Then, add Chloroform, mix them well, wait at RT for 3 minutes before centrifuge.-->Still did not work.
Maybe because the size of the pellet was so small that it was not possible to "resuspend" it after it was frozen with QIAzol.
dear, I am doing the same procedure but failed so far, can I get your protocol ? I could not find a clear protocol for RNA extraction from mouse splenocytes.
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