qPCR from cell pellets

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Amtekoth
Amtekoth's picture
qPCR from cell pellets

Hi,

I have small frozen mammalian cell pellets (10-10000 cells) in a few uL of leftover media. I'm planning on doing some qPCR on the DNA using BioRad's iQ Sybr Green. I'm using 2 different sets of primers.

My plan is to resuspend the pellets in 100uL cold H2O and distribute 20uL x 4 wells into the plate wells with my master mixes. Then just hot start the reaction. My thought is that the starting 94C will break open the cells and inactivate DNAses & proteinases enough to start the reaction without first isolating the very tiny amounts of DNA.

Will this work?

Should I be adding something else to help it work?

Thanks for any advice.

Ed

Fraser Moss
Fraser Moss's picture
Ed

Ed

You should look at this Kit

CellsDirect™ One-Step and Two-Step qRT-PCR Kits
Sensitive and specific, real-time qPCR results directly from cells

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-and-Expression-Profiling/qRT-PCR/Direct-from-Cells.html

Robust results - Real-time detection of targets from a single cell to 10,000 cells

Amtekoth
Amtekoth's picture
Awesome,

Awesome,

Thanks! I will investigate this further and see if I can afford the kit!

Ed

Ivan Delgado
Ivan Delgado's picture
Hi Ed,

Hi Ed,
Did you test your experiment of treating cells at 94oC to perform qPCR directly from cells? I ask because the kits from Invitrogen (CellsDirect qRT-PCR kits) and Applied Biosystems (Cells-to-Ct kits) are very expensive.
I have experience running standard PCR directly from lyzed tissues (Sigma's REDExtract-N-Amp kit; and Finnzymes DNARelease reagent), but not much, and not very positive, qPCR experience in this regard.
Regards

Amtekoth
Amtekoth's picture
 I tried a kit from Promega

 I tried a kit from Promega (DNA IQ) on my small samples and recovered enough DNA to get my PCR reactions to work fine!  Saved me!
 

Ivan Delgado
Ivan Delgado's picture
Thanks for the info. Cheers

Thanks for the info.
Cheers

shareef
shareef's picture
Thanks Ivan for that post

Thanks Ivan for that post after reading thru those posts I found out someone in our lab had a similar problem.  I told them about these posts and it seems to have helped them out.  Thanks

Cheryl020
Cheryl020's picture
You should try Neogen's kits.

You should try Neogen's kits. I have used them and they work well

Oct4
Oct4's picture
Do you have a link to the

Do you have a link to the Neogen kit? Their web site seems mainly ELISA-focused.
 
Also does anyone have any experience doing this from cell clumps e.g. tumor spheroids? ideally I would like a one-pot reaction where I could put a single spheroid into a well, lyse, add real-time PCR reagents, and quantitate the number of genome copies (number of cells) present. I have designed some primers to LINE sequences that should have copy number ~1000 per haploid genome, so I would expect sensitivity should be OK. Right now I am counting by hand after trypsinizing and it's a major pain.