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How to design primers to check my candidates in a ChiP assay


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#1 emmet080

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Posted 20 July 2010 - 12:18 PM

Hi all out there

I hope some one can help me out

I'm currently validating hits from an array study to se if they are true targets of my protein (a transcription factor) or not. My PI want me to conduct a ChiP assay only focusing on the most primary candidates.

My question is. My top candidates does not have known promotor regions and my transcriptionfactor does not have a known binding site. So how should I go ahead when I design primers for my PCR? Should I simply estimate that the promotor is 1-2kb after the transcription start site and design primers toward the complete area or is there a simpler method.

all help is appreciated
/emmet080

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#2 Mighty Mouse

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Posted 25 July 2010 - 08:19 PM

I assume you're following up from a ChIP-microarray (i.e, ChIP-Chip)? If so, then you should have access to the sequences which popped up on the array, I would design my primer for your more focused ChIP around those sequences that had the biggest hits.

If you are talking about a cDNA microarray, and you are trying to figure out if your protein is regulating the transcription of a particular gene that came up from that, then I would start with using the area immediately upstream from the 5'UTR as a starting point. Keep in mind that you might have alternate transcripts and alternate transcriptional start points and/or enhancers/represser elements many kb's upstream from your gene. Anyway...good luck!

MM
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#3 emmet080

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Posted 27 July 2010 - 04:19 AM

I assume you're following up from a ChIP-microarray (i.e, ChIP-Chip)? If so, then you should have access to the sequences which popped up on the array, I would design my primer for your more focused ChIP around those sequences that had the biggest hits.

If you are talking about a cDNA microarray, and you are trying to figure out if your protein is regulating the transcription of a particular gene that came up from that, then I would start with using the area immediately upstream from the 5'UTR as a starting point. Keep in mind that you might have alternate transcripts and alternate transcriptional start points and/or enhancers/represser elements many kb's upstream from your gene. Anyway...good luck!

MM


thank you

#4 Froza

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Posted 01 August 2010 - 06:26 AM

maybe a couple of primers every hundreds of basepaires and some may be the one(s)

#5 Radish

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Posted 04 August 2010 - 10:41 AM

Hi all out there

I hope some one can help me out

I'm currently validating hits from an array study to se if they are true targets of my protein (a transcription factor) or not. My PI want me to conduct a ChiP assay only focusing on the most primary candidates.

My question is. My top candidates does not have known promotor regions and my transcriptionfactor does not have a known binding site. So how should I go ahead when I design primers for my PCR? Should I simply estimate that the promotor is 1-2kb after the transcription start site and design primers toward the complete area or is there a simpler method.

all help is appreciated
/emmet080

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If you are chiping transcription factors you can design internal controls...
Usually I design a primer set flanking the transcription factor binding site (and you expect to see enrichment) and a primer set 2kb upstream the TF bs and a set 2kb downstream de TF bs (usually you don't see any enrichment for these).
As a positive control I choose a well caracterized gene that is transcribed by my transcription factor and that is constitutively active (or at least that is known to be transcribed in your cell type).




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