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Poor data quality using cultured cell in aCGH

aCGH microarray DNA extraction

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10 replies to this topic

#1 kwhk

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Posted 04 February 2014 - 08:27 PM

I use Agilent platform to preform my aCGH experiment. I don't know why the DLR Spread is always evaluated (>0.3) when using primary cultured cell for aCGH (1M, 4x44K and 8x60K). I checked the DNA quality using Nanodrop 2000 and Bioananlyzer DNA7500. Concentration> 100ng/ul, 260/280>1.8 and 260/230>1.8, no smear was found in Bioanalyzer profile. The labeling efficiency fall in the normal range like other successful array. I am very frustrated since all QCs look perfect but the final DLR spread is evaluated and the data quality is poor. Pls help!



#2 mdfenko

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Posted 05 February 2014 - 05:05 AM

you may not be getting proper distribution of the sample in the sandwich.

 

when setting up your sandwich, do you prevent the sample from touching the gasket before you close it?

 

do you ensure full coverage by rotating the sandwich?

 

can you post a scan?


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#3 kwhk

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Posted 05 February 2014 - 07:22 PM

Thx for your reply. I attached the QC metric in PPT format here. I think it may not due to the sandwich problem as I use 4x44K and 8x60K and only the array from primary cell culture sample type will have this kind of problem (ie high in DLRS). Other samples on the same chip will have DLRS between 0.20 to 0.13. However, high DLRS doesn't happen to all DNA extracted from primary cultured cell. In our experience, it has around 25% of failure rate. Other sample types will only have 10% - 5% failure rate which mostly due to the very limited sample input.

 

 

Attached File  QC Report of Cultured Cell.pptx   750.32KB   69 downloads



#4 mdfenko

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Posted 06 February 2014 - 05:03 AM

your 260/230 ratio is a little low (protocol says it should be >2.0)

 

how much dna are you using for each sample (the protocol calls for >=500ng for 4X array and >=250ng for 8X array)?

 

have you tried switching dyes?


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#5 kwhk

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Posted 06 February 2014 - 05:33 PM

For this sample, I used 8x60K array and the input amount was 250ng. The 260/280 is 1.92 and 260/230 is 2.32, concentration is 224.6 ng/ul. The sample was diluted 10 times before processing. No dye swap was preformed.



#6 mdfenko

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Posted 08 February 2014 - 04:06 PM

250ng is the suggested minimum sample. can you try using more?


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#7 mdfenko

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Posted 10 February 2014 - 04:58 AM

looking at the troubleshooting section of the protocol manual, results can be affected by the presence of rna in the sample.

 

you may be able to improve results if you treat with rnase (and re-evaluate the concentration of gdna).


Edited by mdfenko, 10 February 2014 - 04:58 AM.

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#8 kwhk

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Posted 11 February 2014 - 05:38 PM

Thx for your reply mdfenko. I almost did what I could to to optimize the protocol. For DNA input, I tried 200, 250, 300 and 500ng but nothing change with the poor DLRS. I use Qiagen DNA extraction kit and applied 8ul RNase A (100mg/ml from invitrogen) treatment with proteinase K for >30 mins before extraction start. I also suspect that the RNA contamination may contribute to this problem. Do you think it is good to treat the gDNA with RNase A again after extraction? Do you have the suggested protocol on that?



#9 mdfenko

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Posted 12 February 2014 - 04:58 AM

if rna contamination is the cause of your problem then some rna is surviving the initial treatment with rnase. under those circumstances additional treatment with rnase after purification would be warranted.

 

i would just add rnase to the genomic dna solution, incubate for ~1 hr (depending on ratio of rnase to nucleic acid) at room temperature or 37C. then repurify the genomic dna (phenol/chloroform/iaa extraction or column purification, your choice).

 

you may also want to try increasing the incubation time with rnase in your initial purification.


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#10 kwhk

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Posted 12 February 2014 - 05:21 PM

I would prefer column purfication. Do you have any suggestion on the procedures? or I just need to follow the Qiagen DNAeasy extraction kit, ie RNAse A + PK (1 hr;37oC) -> AL (10mins.;56oC) -> 100% ethanol  -> AW1 -> AW2 -> Elute in AE? Should I skip PK and AL steps?



#11 mdfenko

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Posted 13 February 2014 - 05:01 AM

since all you want to do is eliminate the rna, you should just treat with rnase. there is no special procedure, just add rnase, incubate and purify.


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