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Wacky BioAnalyzer Results

Agilent bioanalyzer RNA total RNA troubleshoot

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12 replies to this topic

#1 RueTheDay

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Posted 04 January 2012 - 02:37 PM

Hi All

Has anybody ever seen anything like these?
I'm asking this question in as many forums as possible since nobody around me seems to have any clue what is going on - even the techs at Agilent say they've never seen traces like these.

They are total RNA isolations. I have troubleshot this issue several times, thought that I'd solved the problem, only to have it crop back up again and be solved again.

Please assume that i am following the Agilent manual fairly closely at this point and have even added steps suggested by Agilent techs from my previous troubleshooting sessions.

Earlier solutions that seemed to have fixed the problem before include extra time to equilibrate all reagents to room temp, vortexing the prepped chip at 2000 rather than 2400, and allowing extra time for the electrodes to fully dry after RNase-free water wash. The same samples that gave me problems, would then read just fine once each of these changes in protocol were made. I have also tested for vibration from a nearby centrifuge and shearing of the gel by high speed centrifugation.

Several other people, from several other labs are using this same machine [often times in between 2 of my runs] and nobody is getting anything even close to this. The issue is absolutely something I'm doing wrong with my prep.

The attached photos are recent, from one of four runs using the same samples. No sample gave the same results twice in any of the four runs. Clearly the samples are not the problem.

HELP!

Thanks

Attached Thumbnails

  • 120103-2.JPG
  • 120103-2_GEL.jpg


#2 DRYTCYV

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Posted 04 January 2012 - 04:03 PM

And the ladder? Ok? Is it Pico or Nano Chip?

#3 jpopesku

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Posted 04 January 2012 - 05:07 PM

Is it possible that the sample is not being loaded properly into the bottom of the chip well giving you wonky results due to air bubbles?
It might be worth having someone else try loading your samples into the wells, in case it's "operator error" (happens to the best scientists too!)...

As DRYTCYV says, how about the ladder?

#4 DRYTCYV

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Posted 05 January 2012 - 09:25 AM

Hello,

Like jpopesku says it´s very important the sample dispensing. Should be in the bottom of the chip and you need to stop in the first position in the dispensing movement.
Checking again you photos, i confirm that your ladder is not in good shape :) .

Some important points (sorry if you think this is obvious, but is the best way to find errors):

- Put samples and ladder in hot block (72ºC) 2 min than direct to ice.
- i prepare my gels in the day that i use, so, i had the 0.5 ul of dye in 33 ul of gel -> vortex and 10 min centrifuge at max.
- After load the first well from the gel->syringe > attention to pull softly to the 1ml mark then open.
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- put again in 2400 in the chip vortex, i think that´s not the problem.

- Check if your ladder´s peak is in the correct position

The last problem here, was a problem in the syringe, and create problem in the gel and change everything. But the peaks were shift from the suposed position.

..........try again! :)

#5 RueTheDay

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Posted 05 January 2012 - 11:27 AM

Hi Guys!

Thanks so much for responding!

So yeah the ladder looks just as ugly, and the trace varies from run to run just like the other samples, so it's definitely a loading/read issue

No offence regarding the obviousness of suggestions - the solution will almost definitely be some stupid, obvious little thing I'm doing wrong...

- These are Nano chips
- I am heat denaturing my samples and ladder as described
- I do prepare my gel on the day of the run [though in the volumes suggested by the manual] - curious about the 10min centrifuge - how necessary do you all think the full 10 min spin is? DRYTCYV, you say you do it at full speed, but I've been told that the lower speed recommended is based on the possibility if shearing the gel [this was a parameter I attempted to test for - to no avail]
- I am being quite careful drawing up the syringe after priming
- the 2400 rpm vortex was changed to 2000 when we thought that the problem could be liquid on the edges causing current leakage between electrodes; i think you're right tho - I should probably switch back

I'll keep plugging away - thanks again!

#6 DRYTCYV

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Posted 05 January 2012 - 01:56 PM

Hello

The 10 min at full spin (in my centrifuge 14000rpm) if after vortex the dye and the gel, not in the spin filter. It´s in the manual.
It´s true, putting the chip in the vortex it´s crazy, seems like everything goes jump off Posted Image .

#7 WSN

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Posted 06 January 2012 - 08:09 AM

You did all right except for the input amount is out of the range, most likely, over the up limit. Adjust the amount using NanoDrop spectrum read. Good luck!

#8 DRYTCYV

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Posted 06 January 2012 - 04:49 PM

Yes, this is true the level of fluorescence is low. But if ladder is bad....something is wrong.
But like WSN says, adjust the amount or try it in an Pico Chip (50-5000pg). But no ladder no game! :)

#9 RueTheDay

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Posted 09 January 2012 - 11:39 AM

my samples generally come out to be anywhere from 100ng/µl to 1µg/µl - which should be in the Nano chip's range - and again, I have in the past 'solved' the problem and the samples which were giving me problems then gave excellent results.

This has to be a stupid prep issue on my part

Edited by RueTheDay, 09 January 2012 - 12:26 PM.


#10 RueTheDay

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Posted 09 January 2012 - 11:48 AM

Let me add these picts

These are the exact same samples, later run. This is new ladder, freshly denatured, from the above pic.

Attached Thumbnails

  • 120105.jpg
  • 120105_GEL.jpg


#11 pDNA

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Posted 09 January 2012 - 02:14 PM

could be due to genomic DNA contamination that causes rise and fall in baselines ...prefered in line 7-11. These baseline inregularities are due to the presence of longer ssDNA fragments of gDNA.

How did you isolate your RNA? ...is a gDNA contamination possible? Have you run your sample on a agarose gel before?

Regards,
p

#12 DRYTCYV

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Posted 09 January 2012 - 03:15 PM

If you had problems to get a good ladder profile, than you got a general problem.
Your ladder profile sugest some problems in the gel, or the pressure.

Next idea: The Syringe clip should be the upper position (Nano and Pico Chips)

#13 RueTheDay

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Posted 11 January 2012 - 10:32 AM

Yep, the clip is in the upper position. I was concerned that pressure may the issue, but when the clip is released [after 30 sec], the plunger shoots back up past the 0.9 marker before i gently pull it the rest of the way up.





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