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RNA-IP


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3 replies to this topic

#1 protein2

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Posted 05 August 2009 - 07:42 AM

I'm trying to perform an RNA-IP (immunoprecipitate a protein of interest and extract the mRNA bound to it). Whenever I lyse my cells with my modified RIPA buffer, I get fluffy white precipitate in my lysate that looks like genomic DNA. Could this contain RNA as well? If I pellet the lysate and use the supernatant for the RNA-IP, could I expect to obtain any RNA from my IP-ed samples?

The composition of my buffer is:

50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors

Thanks!

#2 drjcroberts

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Posted 05 August 2009 - 10:37 AM

I'm trying to perform an RNA-IP (immunoprecipitate a protein of interest and extract the mRNA bound to it). Whenever I lyse my cells with my modified RIPA buffer, I get fluffy white precipitate in my lysate that looks like genomic DNA. Could this contain RNA as well? If I pellet the lysate and use the supernatant for the RNA-IP, could I expect to obtain any RNA from my IP-ed samples?

The composition of my buffer is:

50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors

Thanks!



hi,

i have several papers on this, your RIPA buffer will indeed be removing chromatin (and nucleii)

there are published methods on this, its known as RIP or CLIP

J

#3 protein2

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Posted 05 August 2009 - 01:48 PM

Thank you for your reply - but I'm concerned about whether the pellet from my lysate that I will discard (which contains chromatin and insoluble nuclear material) also contains the RNA that I need. Although I have read the published protocols on RIP, they never mentioned anything about losing RNA during the lysis process, so I was wondering if this was a common occurrence.

hi,

i have several papers on this, your RIPA buffer will indeed be removing chromatin (and nucleii)

there are published methods on this, its known as RIP or CLIP

J



#4 Penguin

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Posted 06 August 2009 - 05:30 AM

I'm trying to perform an RNA-IP (immunoprecipitate a protein of interest and extract the mRNA bound to it). Whenever I lyse my cells with my modified RIPA buffer, I get fluffy white precipitate in my lysate that looks like genomic DNA. Could this contain RNA as well? If I pellet the lysate and use the supernatant for the RNA-IP, could I expect to obtain any RNA from my IP-ed samples?

The composition of my buffer is:

50 mM Tris-HCl pH 7.5
150 mM NaCl
0.5% NP-40
0.5% Sodium Deoxycholate
Protease, RNase inhibitors

Thanks!


Cellular RNAs are much shorter than the genomic DNA and so should not precipititate out in the same way. I have used RIPA for RNA-IPs and have managed to see my RNA of interest by RT-PCR,
P




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