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Microarray hybridization problem


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#1 dkmishra

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Posted 08 January 2010 - 07:07 PM

Hi,
I am new in microarray hybridization experiments. I am getting empty streaks (lines) during scanning of my hybridized oligo array. Any one can help me in diagnosing the problem and solution for this.

Thanks

#2 pDNA

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Posted 10 January 2010 - 12:21 AM

What do you mean by empty streaks?
What equimpent do you use?

Regards,
p

#3 dkmishra

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Posted 11 January 2010 - 08:55 PM

Empty streaks means no hybridization in area that looks like a line. We use Genepix Pro 400B for scanning.
Thanks

#4 gogreen

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Posted 12 January 2010 - 08:26 AM

Lines seen on arrays are mostly washing artifacts..If u see lines, it can be because of the wash buffers drying up during the time you lift the arrays from the wash buffer. if you pull out the arrays from the wash buffer in a single smooth movement, most of these can be avoided...(I presumed you do a manual wash of the arrays)...

what arrays do u use? what buffer do u use?? Do u wash the slides manually?? mentioning all these would make it easier to answer !

gg

Edited by gogreen, 12 January 2010 - 08:52 AM.


#5 mdfenko

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Posted 12 January 2010 - 08:34 AM

do you use gasket slides? if so, then are you sure it was placed properly?

picture would help.
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#6 kaveh

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Posted 12 January 2010 - 09:41 AM

Hello,
That's probably because you do your washes manually. Make sure you dry the slide (preferably by a slide spin) immediately after the last wash. You should avoid letting the wash buffer to dry out on the slide. Also, be careful not to touch the slide by your gloves or any plastic ware (always hold the slide along the edges). One more thing, keep the light away to avoid getting photo beaches.

k

#7 gogreen

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Posted 12 January 2010 - 11:37 AM

A brief wash (10-30 sec) in acetonitrile would help reduce the washing artifacts

#8 mdfenko

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Posted 14 January 2010 - 11:38 AM

i have seen where a reader malfunction has caused streaks in the image.

i reset the machine (turned it off then back on) and that corrected the problem. if yours is the same then try resetting. if that doesn't work then it may be time to call for repair.
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#9 array75

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Posted 18 January 2010 - 08:05 AM

i have seen where a reader malfunction has caused streaks in the image.

i reset the machine (turned it off then back on) and that corrected the problem. if yours is the same then try resetting. if that doesn't work then it may be time to call for repair.



Hi,
depends, if these lines/ streaks are regular, it may be caused by a defect scanner. The 4000B is pretty robust - the detection system is going from left to right. If the lines are exactly within this pattern, the scanner needs some maintenance.
Simple test - scan a normal microscope slide (there is always some fluoro-stuff on these slides) - if these empty lines appear with the same pattern as well - save your loose cash and call Axon TechSupport...


If you see these artefacts in different orienations, it seems more to be a an handling issue. What kind of array and what kind of hyb-chamber do you use? Lifter/ Coverslips? Could be related to scratches during the washing process.
Real washing artefacts look very cloudy with no clear lines.

For finding the problem, much more description is needed, especially for the proportion of the lines. Image would help as well.

Edited by array75, 18 January 2010 - 08:07 AM.





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