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Old 06-12-2012, 01:42 AM   #1
carotylene
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Location: italy

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Default strange bioanalyzer results

I am getting crazy with this RNA extracted from marine copepods! We used different extracting methods and kit, but always get the same bioanalyzer results: a drop off after the 18S peak. The technician said the ladder is degraded and that there can be salt contamination causing the dop. But I doubt it. Why always at the same time? Any suggestion?
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Old 06-12-2012, 05:45 AM   #2
pmiguel
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If the ladder is degraded that is a bioanalyzer run issue. Looks to me like the bioanalyzer operator loaded the ladder well with one of the samples in your right 4 lanes.

If you are sure that this is not an operator mistake, then my second guess would be massive contamination of the pin (electrode) set with one of your samples or samples from an earlier run of the instrument.

My third guess would be that the chip capillaries are defective, but I guess that is unlikely.

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Phillip
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Old 06-12-2012, 05:56 AM   #3
carotylene
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Quote:
Originally Posted by pmiguel View Post
If the ladder is degraded that is a bioanalyzer run issue. Looks to me like the bioanalyzer operator loaded the ladder well with one of the samples in your right 4 lanes.

If you are sure that this is not an operator mistake, then my second guess would be massive contamination of the pin (electrode) set with one of your samples or samples from an earlier run of the instrument.

My third guess would be that the chip capillaries are defective, but I guess that is unlikely.

--
Phillip
Dear Phillip,
you are totally right for the right 4 lanes, but it is the other way round, the operator loaded the ladder also on those 4 lanes to check it.
As for your other guesses, I will discuss it with the operator. We are still waiting for the company operator to come!
I'll let know the development.
Many thanks for your reply!
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Old 06-12-2012, 06:29 AM   #4
pmiguel
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Okay, I get it.

Yes your samples also look degraded.

When is the last time you washed the pins? That is, taken them out and sonicated them and/or otherwise scrubbed them to clean them? (Agilent has protocols available.)

We do that about once a week. Down side is that if even the slightest moisture remains on the pin set, the next run will be ruined. So we try to do the washing just before a weekend so the pins can dry for a couple of days. Otherwise you might have to do an ethanol rinse followed by a few hours in a vacuum to remove all moisture from the pins.

Also, many bioanalyzer facilities keep different pin sets for RNA and DNA. DNA is frequently contaminated with RNAse, so you really don't want to do a DNA chip and follow it with an RNA chip. The little RNAzap wash wells can only do so much.

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