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Gene expression assay with small cell number


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#1 ixsix

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Posted 07 May 2013 - 08:44 AM

Hi all,
I am new in this so any suggestion will be really helpful.

I want to get gene expression profile (miRNA as well) with flow sorted cells. However, since the rarity of my interested cells, I can only get about 10 to 30 thousand cells. So, which method is good for me to get the gene expression profile with these cells? Is there specific kit or related product I can choose? Both microarray and RNAseq can be considered if work for me.

Thank you guys.

Dan

#2 Tabaluga

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Posted 07 May 2013 - 08:57 AM

How much RNA do you approximately get out of this amount of cells ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 ixsix

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Posted 07 May 2013 - 10:44 AM

How much RNA do you approximately get out of this amount of cells ?


I can get about 200-500ng RNA extracted from these cells.

#4 Winegarden

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Posted 12 July 2013 - 11:15 AM

200 to 500 ng of RNA is quite a bit actually now a days. With these amounts of material the microarray methods are probably still more robust (can go down to single digit nanograms for mRNA expression).

Illumina, Affymetrix and Illumina can all handle down to 200 ng quite easily. For Illumina even 50 ng will work if you are careful. If you need to go further down kits from NuGen will allow you to go to 2-5 ng easily (at a cost of course). We've had success going down to 100 pg in some cases with various amplification strategies.

For RNAseq there are several kits that say they can go down to 100 ng. We've not yet tried this. All our RNA seq has been from 1 µg. So I cannot comment on how well they work (yet).

For miRNA - I'd seriously consider NanoString. 100-200 ng will get you a full profile of 800 human (or 580 mouse) miRNAs. Again we've had good experience with this.

We do a lot of work from Sorted cells. The main issues are making sure you do the sorting in such a way that you don't induce a lot of changes in expression. As long as the RNA looks decent (good RIN) the arrays will work fine. If the RNA is degraded you can try Illumina DASL (also down to 100 ng without issue). Nanostring works great on degraded RNA.




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