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Old 08-21-2013, 11:27 AM   #1
Hybridizationx
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Default Covaris FFPE extraction Kit using AFA tubes?

Hey has anybody tried the new Covaris kit for DNA extraction & purification with the AFA tubes? Any luck?

http://covarisinc.com/products/ffpe-extraction/

They use AFA to dissociate the paraffin, instead of xylene, followed by protease K and RnaseA digestion. After this, one can proceed to purification with a column. Covaris claimed that very large DNA fragments could be obtained by this method, peaking at 8kb.
Alternatively, following the digest, one can shear the DNA directly in the same AFA tube to 200-400bp, and then purify. Samples are ready for NGS sample prep.

This seems tempting because we have been using the traditional method to extract DNA with xylene. Gel/bioanalyzer show that DNA is highly fragmented (as everyone else is observing), being a smear ranging from 800 to very short. It would be nice to omit the harsh organic steps to get more intact DNA.
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Old 09-04-2014, 06:05 AM   #2
lomoens
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I'm also interested! Did you have the chance to test this system?
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Old 09-09-2014, 05:16 AM   #3
FWOS
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I have been using the kit for about 6 months and it seems to work well. The protocol is fast and limits the amount of heat needed by only heating up for the ProtK treatment (compared to other preps i.e.: qiagen, etc… with require 2+ hours in a heat block for de-paraffinization and protK

Overall i am very pleased! As a side note - the prep results in total nucleic acid, so you can treat half with Rnase and the other half with DNase if you want DNA and RNA.
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Old 09-09-2014, 05:30 AM   #4
lomoens
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Thank you so much for your answer! Sounds good...

just two more questions:

- what about the yield? comparable/lower/higher than qiagen?
- about the workflow: the system seems to use kind of tiny microtubes and caps. I was just wondering if those are practical to work with when one has a lot of samples to process?

Thanks again!

Lotte
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Old 09-09-2014, 06:43 AM   #5
FWOS
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Hi,

One of the reasons we moved forward with Covaris was the higher yields for both DNA and RNA. (Compared to qiagen all prep FFPE; Kapa Express extract and 5 other kits we tested. The only thing I would worry about would be losing miRNA in a column cleanup etc… That can be easily remedied if you are looking at miRNA, just split the sample after ProtK, DNase treat half and run the sample through a RNAeasy qiagen column at the end.

Side note*: RNA yield was higher than DNA yield not matter the extraction type which makes a lot of sense.

Hope this helps.

Best,
Frank
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Old 09-10-2014, 02:31 AM   #6
nucacidhunter
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Quote:
Originally Posted by lomoens View Post
- what about the workflow: the system seems to use kind of tiny microtubes and caps. I was just wondering if those are practical to work with when one has a lot of samples to process?
The tubes are easy to work with. If you have a lot samples you can use E220 or LE220 systems which can process batch of 96 samples individually or in a group of 8, respectively. Here is a link to their web page: http://covarisinc.com/products/afa-u...tion/l-series/
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Old 09-16-2014, 07:50 AM   #7
FWOS
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We use the LE220 and can run 24 samples at a time. The tubes are larger than regular shear tubes, so the adapter plate can only handle 24 per run. Not too bad to work with the tubes since they have stoppers and can be labeled upfront. Columns are never fun when working with tons of samples, but the extraction could probably be automated to some level if you want to spend the time and money. I think it is far more user friendly and much less complicated than the competition (plus it minimizes damage by deparaffinizing without adding additional heat).
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Old 12-01-2014, 04:30 AM   #8
nr7823
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Default Siemens TPS vs Covaris LE220

Hello,

Has anyone had any experience using either the Covaris LE220 (truXTRAC workflow) or the Siemens TPS system for automated DNA (or RNA) FFPE extraction? Any advantages to one over the other? It seems like the Siemens has been out for some time vs the covaris application. My application is for targeted NGS oncology testing
Thanks very much!
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