Nimblegen CGH array QC issues - (Feb/26/2013 )
I'm new to the whole microarray scene and I'm having real issues with getting my arrays to pass QC. I'm using the Nimblegen CGH DNA arrays and they have an optional Loading and hybridisation control which is causing me grief. This is basically the proxy measure for either a duplication or deletion event seen in the samples, and also showing how well the hybridisation and labelling went. I've checked the DNA quality of my samples before hand and they're all fine and not degraded. And I'm being careful with keeping the labelled samples covered in foil and away from light, which the rep told me could affect the QC. However, my samples are still not passing QC after I've tried again and being careful with light exposure and etc.... Does anyone have any ideas as to what else I can do or am doing wrong that might help?
i use agilent microarrays so i'm not sure if this applies to your situation but...
if you are using cy dyes, they are ozone sensitive (especially cy5). after hybridization and washing we coat with a stabilizer provided by agilent (now, they also have covers that can be used instead of stabilizer, we still use stabilizer).
We don't have a stabliser for our arrays. We just wash, dry and scan the array. The funny thing is that the AC drop outs isn't across the whole array, just random samples here and there, which is puzzling.
if the problem is being caused by ozone then you may want to try a shield (agilent sells a physical shield besides the stabilizer). the physical shield is decribed here. the stabilization and drying solution is described here.
are you sure that you're draining the arrays properly? i found that if i didn't allow the arrays to drain away from the label, then i would get uneven backgrounds.
How do I know if the problem is caused by ozone?
As for proper draining... I swish it quite a few times in the initial wash solution before popping them into the slide containers to wash. I've basically done everything like the person who taught me and she has no or very little issues. The only difference is the DNA samples. As I wasn't provided with enough DNA to re-amplify, I've had the samples rehybridised by the person who taught me (so the labelled DNA is the same as what I have used), and some samples that did not pass QC passed the second time around and the ones that passed QC previously failed the second time. I'm very very puzzled by this.
the cy5 will decompose and you will see the fluorescence reduced or nonexistent.
i found artifacts if i didn't drain away from the label. do you have the washes stirred gently?
Ok. We basically strip away the plastic chambers that keep the arrays separate from each other on the slide while it's in one of the wash buffers and once that's off we swish it around in there for a few seconds. Then the slide is transferred to a slide washing container with another wash buffer and this is shaken vigorously for 2 mins. This process is repeated twice more with different wash buffers before it's spun dry and scanned.
the procedure for agilent arrays is a little different. after separating the array slide from the gasket slide in wash buffer, the array is washed in the same buffer (with triton x-114 if necessary to reduce background) for 1 minute with gentle stirring. it is then washed in another wash buffer (with triton x-114 if necessary) for 1 minute at elevated temperature with gentle stirring. air dried if ozone is not a problem or soaked in acetonitrile for 1 minute (with gentle stirring) then stabilizer for 30 seconds (with gentle stirring) and air dried.
vigorous handling is discouraged.