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Please help microarray beginner - (Oct/26/2010 )

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.

-evilid-

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.

-Clare-

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.

-evilid-

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.

-Clare-

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.

-evilid-

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.

-Clare-

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.

-evilid-