Please help microarray beginner - (Oct/27/2010 )
Hell0, everyone.
I am trying to troubleshoot my DNA microarray.
Any help will be appreciated.
I am trying ChIP-on-chip.
I am using Agilent system and following the instruction of the company without modification.
I tried it once with a person who has lots of experience and it was pretty much successful.
However this time I tried on my own and it seems to be failed.
Basically I think I got very weak signal.
Previously I had nice green and red dots on black background after scanning.
This time I got weak and grainy dots on very strong green background.
I would like to ask two questions.
1. Why do I get green background? If my labeling or hybridization didn't work well, shouldn't I get very dim dots on black background. Why green background? Does the scanner have a sort of compensation function? Does it try to get as much as signal by increasing laser power when the signals are weak?
2. How would I improve my labeling? More specifically, how would I make sure labeling is adequate? The instruction says that I need at least 2 pmol/ul for Cy5 and 3 pmol/ul for Cy3. I got 5.9 and 8.0, respectively. Does this mean that I got too much incorporation or that I have lots of unlabeled dye on my samples due to insufficient washing?
Thank you very much.
Hi there
I have done hundreds of chip-chips with Agilent slides - can you tell us your entire protocol (from labelling to hybridisation to washing)?
If you are using the Agilent scanner and software, I know that if you do have very weak signals, the background colours can look wierd.
In terms of improving your labelling, I would need to see you protocol first
Regards
Clare
evilid on Wed Oct 27 06:00:14 2010 said:
Hell0, everyone.
I am trying to troubleshoot my DNA microarray.
Any help will be appreciated.
I am trying ChIP-on-chip.
I am using Agilent system and following the instruction of the company without modification.
I tried it once with a person who has lots of experience and it was pretty much successful.
However this time I tried on my own and it seems to be failed.
Basically I think I got very weak signal.
Previously I had nice green and red dots on black background after scanning.
This time I got weak and grainy dots on very strong green background.
I would like to ask two questions.
1. Why do I get green background? If my labeling or hybridization didn't work well, shouldn't I get very dim dots on black background. Why green background? Does the scanner have a sort of compensation function? Does it try to get as much as signal by increasing laser power when the signals are weak?
2. How would I improve my labeling? More specifically, how would I make sure labeling is adequate? The instruction says that I need at least 2 pmol/ul for Cy5 and 3 pmol/ul for Cy3. I got 5.9 and 8.0, respectively. Does this mean that I got too much incorporation or that I have lots of unlabeled dye on my samples due to insufficient washing?
Thank you very much.
Hello, Clare.
I am EXACTLY following Agilent Mammalian ChIP-on-chip Protocol version 10.11.
We basically use this as a working protocol without any modification (reagent companies, sonicator brand, amounts of reactions and so on.)
I am doing 4x18K custom chips.
What I am suspecting is inefficient removal or unincorporated dyes or inefficient blocking.
However I do not have expertise on this matter.
For example I am not sure whether I can wash labeled samples more extensively or not.
Also I am not sure how I can check labeling efficiency other than O.D.
Thank you for your help.
Clare on Thu Oct 28 14:40:18 2010 said:
Hi there
I have done hundreds of chip-chips with Agilent slides - can you tell us your entire protocol (from labelling to hybridisation to washing)?
If you are using the Agilent scanner and software, I know that if you do have very weak signals, the background colours can look wierd.
In terms of improving your labelling, I would need to see you protocol first
Regards
Clare
evilid on Wed Oct 27 06:00:14 2010 said:
Hell0, everyone.
I am trying to troubleshoot my DNA microarray.
Any help will be appreciated.
I am trying ChIP-on-chip.
I am using Agilent system and following the instruction of the company without modification.
I tried it once with a person who has lots of experience and it was pretty much successful.
However this time I tried on my own and it seems to be failed.
Basically I think I got very weak signal.
Previously I had nice green and red dots on black background after scanning.
This time I got weak and grainy dots on very strong green background.
I would like to ask two questions.
1. Why do I get green background? If my labeling or hybridization didn't work well, shouldn't I get very dim dots on black background. Why green background? Does the scanner have a sort of compensation function? Does it try to get as much as signal by increasing laser power when the signals are weak?
2. How would I improve my labeling? More specifically, how would I make sure labeling is adequate? The instruction says that I need at least 2 pmol/ul for Cy5 and 3 pmol/ul for Cy3. I got 5.9 and 8.0, respectively. Does this mean that I got too much incorporation or that I have lots of unlabeled dye on my samples due to insufficient washing?
Thank you very much.
Hi
Are you using the Agilent oven for your hybs? I never got a nice result using their oven, so switched to an Implen SlideBooster. I'll read the protocol and see if I can spot any potential issues...
but in the meantime...
What do your DNA samples look like before labelling? Do you use a nanodrop - what are the ratios?
FYI: I used this labelling kit from Invitrogen which gave fantastic results - it's not cheap though
http://products.invitrogen.com/ivgn/product/A10963010?ICID=search-product (BioPrime® Total for Agilent® aCGH)
Clare
evilid on Fri Oct 29 00:39:46 2010 said:
Hello, Clare.
I am EXACTLY following Agilent Mammalian ChIP-on-chip Protocol version 10.11.
We basically use this as a working protocol without any modification (reagent companies, sonicator brand, amounts of reactions and so on.)
I am doing 4x18K custom chips.
What I am suspecting is inefficient removal or unincorporated dyes or inefficient blocking.
However I do not have expertise on this matter.
For example I am not sure whether I can wash labeled samples more extensively or not.
Also I am not sure how I can check labeling efficiency other than O.D.
Thank you for your help.
Hi, Clare.
Yes, we do have Agilent oven.
Our system was purchased as a package from Agilent.
I will look into another option on this matter.
My DNA samples look okay on agarose gel after LM-PCR.
Nice smearing at around 300-500bp.
Amounts were around 4-5 ug.
After labeling, Input was 0.31ug/ml (5.9pmol/ul), IgG contol was 0.35ug/ml (8.0pmol/ul) and my IP sample was 0.3ug/ml (8.5pmol/ul) according to Nanodrop measurement.
Thank you.
Hello
Hmm...from what you have said, it seems like everything is ok with your DNA - do you check your results vis real time PCR before you amplify and label?
Here's my protocol if it's any help:
http://amplispeed.com/advalytix/images/downloads/Implen_ApplicationNoteChIP-on-chiponSlideBooster.pdf
I wonder if your slides look anything like the crap one which is on the PDF?
Clare
evilid on Mon Nov 1 01:13:47 2010 said:
Hi, Clare.
Yes, we do have Agilent oven.
Our system was purchased as a package from Agilent.
I will look into another option on this matter.
My DNA samples look okay on agarose gel after LM-PCR.
Nice smearing at around 300-500bp.
Amounts were around 4-5 ug.
After labeling, Input was 0.31ug/ml (5.9pmol/ul), IgG contol was 0.35ug/ml (8.0pmol/ul) and my IP sample was 0.3ug/ml (8.5pmol/ul) according to Nanodrop measurement.
Thank you.
Hello, Clare.
I found a new insight with this matter.
Careful comparison with the protocol and my labnote, I found that my hybridization was not ideal.
Basically cotDNA was 1/3 and hybridization solution was 0.33x.
This explains why my slide was all green.
Thank you for your help.
I really appreciate it.
Clare on Mon Nov 1 11:27:21 2010 said:
Hello
Hmm...from what you have said, it seems like everything is ok with your DNA - do you check your results vis real time PCR before you amplify and label?
Here's my protocol if it's any help:
http://amplispeed.com/advalytix/images/downloads/Implen_ApplicationNoteChIP-on-chiponSlideBooster.pdf
I wonder if your slides look anything like the crap one which is on the PDF?
Clare
evilid on Mon Nov 1 01:13:47 2010 said:
Hi, Clare.
Yes, we do have Agilent oven.
Our system was purchased as a package from Agilent.
I will look into another option on this matter.
My DNA samples look okay on agarose gel after LM-PCR.
Nice smearing at around 300-500bp.
Amounts were around 4-5 ug.
After labeling, Input was 0.31ug/ml (5.9pmol/ul), IgG contol was 0.35ug/ml (8.0pmol/ul) and my IP sample was 0.3ug/ml (8.5pmol/ul) according to Nanodrop measurement.
Thank you.