Problematic bioanalyzer results (QC after mRNA capture)

[sbchua.1990]

Did mRNA capture (Dynabead) for my RNA extract samples (mushroom). When run mRNA pico assay for QC, these kind of result appeared. Sometime it work for some of the samples. But repeated assay using same sample seem to produce different result. Contacted field application specialist and made the listed changes but problem persists.

Changes made:

1. washed electrode with RNAzap and consequently with RNase free water and air dried for more than 10 minutes. Even diagnosed for short circuit before run.

2. check priming station for failure. It seem to be working because it bounce normally when the latch was released

3. my sample were treated with DNase from MoBio.

4. diluted my samples.

Attached here are pdf. containing results for last few run.

2100 expert_mRNA Pico_DE13805702_2016-06-27_18-03-51.pdf

2100 expert_mRNA Pico_DE13805702_2016-06-14_17-28-00.pdf

2100 expert_mRNA Pico_DE13805702_2016-06-27_16-40-47.pdf

[DStephens@NuGEN]

Hi sbchua.1990,
We have seen traces such as these when the chip priming/loading was not performed correctly. Did you ensure that the chip priming station plunger was set to the highest notch and the plunger was released after exactly 30 seconds? Was the chip loaded with fresh reagents?

A couple of other possibilities include overloading of the chip and salt or detergent carryover. You noted that you already tried diluting the samples; how much material was used? We typically load 1-5 ng/well onto the Pico chip.

Finally, have you tried an additional clean-up of your samples?

Hope that helps!

[sbchua.1990]

Thanks for reply, I am pretty sure that the chip priming/loading was performed correctly. Reagents are still within expiration date.

My samples concentration should be around 300pg - 1500pg. I just loaded 1 ul without dilution.

No I did not try additional clean-up for my samples.

[nucacidhunter]

The samples most likely are contaminated with residual removal resin. You would need to contact MoBio for cleanup instructions.

[sbchua.1990]

Hi nucacidhunter, did you encounter such problem in the past? Or what make you think so? Thanks for your reply.

[nucacidhunter]

Profile indicates presence of insoluble contaminants which could be protein (residual DNase) or removal resin. I suspect the resin because it can be carried over easily if spinning was not long enough or one tried to take the supernatant without leaving any liquid behind in the tube. There is also possibility of protein protectant carry over which is used to stabilise DNase at RT.