Using Covaris S2 for RNA shearing

shurjo · 05-28-2009, 09:31 AM

Does anyone have any experience using the Covaris AFA technology for
RNA fragmentation? While I'm not sure AFA can be used on RNA, if
anyone has tried it I would appreciate any feedback. The Covaris
website hints that AFA may only work for DNA fragmentation: "Double
stranded DNA (unlike RNA) will shear when exposed to the energy of
AFA".

qdxjcn · 01-06-2010, 08:26 AM

Co ask. Did anyone has the experience for RNA shearing to target size fragment with AFA?

Hamid · 01-08-2010, 10:11 AM

Hi Shurjo,

We have a few customer who have tried a 2-25 minute time course of the setting below to shear RNA:
10%dc,5i, 1000cpb.

A customer generated an average shearing size of 400-500b only after a 2 minute treatment using a concentration of less that 100ng/ul. My suggestion would be to use the setting above and carryout an initial test time course of 2-25 minutes depending on the concentration of the RNA sample you will ultimately shear.

Thank you

hamid

shurjo · 01-20-2010, 02:46 PM

Hi Hamid,

Thanks for the info. These days I routinely use:

10% DC, 5 Intensity, 200 cpb and 75 seconds

for 150ng of polyA RNA in a 120ul volume of H2O in a 6x16mm tube. This gives a nice tight distribution around 200bp every time.

Regards,

Shurjo

nextgen · 02-14-2010, 08:10 AM

Covaris works great for RNA shearing. You can get a very tight range. Just play around with it.

nextgen · 02-14-2010, 08:13 AM

I used 500bp range as this right range for 454 (some mRNA transcripts are in that range.) You can easily get smaller size. But for random or end-to-end cDNa synthesis, 500bp is better.

ik76 · 03-04-2010, 01:27 AM

Hello,
I was just wandering whether anybody have tried fragmenting total RNA using Covaris ( as a replacement for the fargmentation in zink solution recommnded in the Illumina mRNA sample prep protocol). If so, what was the starting concentration and Covaris conditions? Did you have problems with RNA degrading ?
if you have started with purified mRNA, how did you measure the conc ? Will just using Agilent Pico chip work ?

kshankar · 10-22-2010, 09:08 PM

This is the only place I have been able to find some starting conditions for RNA fragmentation using the Covaris S2. Using the conditions that Shurjo mentioned (and some longer times, ie upto 3 mins), we are getting 100-500 range centered around 200 bp. What is the ideal range and size for fragmented RNA for Illumina mRNA-seq? Also, Does anyone know if Covaris has come out with specific settings?

thanks for your help
--K

josdegraaf · 10-25-2010, 05:13 AM

Quote:

Originally Posted by kshankar

This is the only place I have been able to find some starting conditions for RNA fragmentation using the Covaris S2. Using the conditions that Shurjo mentioned (and some longer times, ie upto 3 mins), we are getting 100-500 range centered around 200 bp. What is the ideal range and size for fragmented RNA for Illumina mRNA-seq? Also, Does anyone know if Covaris has come out with specific settings?

thanks for your help
--K

Last time I spoke with them not..

Hamid · 10-29-2010, 01:36 PM

the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
I have attached a sample data kindly provided by a few of your customers.

Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

Thank you

Hamid

04BCH022 · 12-06-2010, 03:22 PM

Quote:

Originally Posted by Hamid

Hi Shurjo,

We have a few customer who have tried a 2-25 minute time course of the setting below to shear RNA:
10%dc,5i, 1000cpb.

A customer generated an average shearing size of 400-500b only after a 2 minute treatment using a concentration of less that 100ng/ul. My suggestion would be to use the setting above and carryout an initial test time course of 2-25 minutes depending on the concentration of the RNA sample you will ultimately shear.

Thank you

hamid

Hello Hamid,

I regularly use the covaris to shear RNA and get a nice peak at around 200bp. I am trying different settings to get that peak at 400-500bp. Are you aware of any settings which kind of shift the "triangle" further away from the marker peak (on the agilent) so that the "triangle's" peak is around 400-500bp.
I have ran a bunch of different sets with varying intensities, time C/B, DC but havent yet found the correct configuration.

Thank You.

Regards.

jaywang · 04-12-2011, 06:26 AM

It looks like that there is always a small peak around 25bps. Are they mature microRNAs?

Quote:

Originally Posted by Hamid

the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
I have attached a sample data kindly provided by a few of your customers.

Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

Thank you

Hamid

RCJK · 09-15-2011, 11:50 PM

I just thought I'd give this thread a bump and see if there are any updates regarding using Covaris for RNA shearing, in particular for 454 libraries? We've recently obtained a Covaris and I'm hoping to try it out instead of using Roche's fragmentation procedure. Thanks!

kshankar · 09-16-2011, 07:42 AM

RCJK
We use the following conditions for mRNA, 120 μl volume, 10% DC, 5 intensity, 200 cpb, 7.5 min and get highly reproducible 150 bp fragmentation peaks. We are using these for Illumina libraries. If you want longer length just shorten the time. In our hands, 30 sec fragmentation gives ~ 500 bp peak, 60 sec ~ 400 bp, 180 sec ~ 200 bp fragments. hope that helps.
-Kartik

RCJK · 09-19-2011, 08:12 PM

Thanks Kartik!

ETHANol · 09-19-2011, 10:55 PM

I've jsut stated using the Covaris for chromatin fragmentation and so far I'm very impressed with the results. The reproducibility between samples is impressive.

What I am wondering is what are the advantages/disavantages of using the Covaris vs. chemical methods for shearing RNA for RNA-seq library preparation?

aleferna · 02-03-2016, 01:21 PM

Quote:

Originally Posted by ETHANol

I've jsut stated using the Covaris for chromatin fragmentation and so far I'm very impressed with the results. The reproducibility between samples is impressive.

What I am wondering is what are the advantages/disavantages of using the Covaris vs. chemical methods for shearing RNA for RNA-seq library preparation?

never got a answer for this, so what is better Covaris or the chemical/heat fragmentation? ?

Hamid · 02-03-2016, 01:29 PM

Physical shearing is better since it is concentration independent, isothermal, easily controllable as compared to chemical methods, and you don't have to deal with the stringent cleanup methods required after chemical/heat fragmentation.

Thank you.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Shearing Chromatin using Covaris	Bonn	Sample Prep / Library Generation	15	08-21-2012 11:28 AM
ChIP - shearing with the Covaris S2	ETHANol	Epigenetics	2	08-10-2012 12:47 AM
covaris shearing range	chaos81	Illumina/Solexa	1	08-14-2011 06:20 PM
shearing question using Covaris	seqgirl123	Sample Prep / Library Generation	3	04-28-2011 11:17 AM
Shearing Amplicons with Covaris	lzembek	454 Pyrosequencing	1	01-05-2011 09:34 AM

05-28-2009, 09:31 AM	#1
shurjo Senior Member Location: Rockville, MD Join Date: Jan 2009 Posts: 126	Using Covaris S2 for RNA shearing Does anyone have any experience using the Covaris AFA technology for RNA fragmentation? While I'm not sure AFA can be used on RNA, if anyone has tried it I would appreciate any feedback. The Covaris website hints that AFA may only work for DNA fragmentation: "Double stranded DNA (unlike RNA) will shear when exposed to the energy of AFA".

10-22-2010, 09:08 PM	#8
kshankar Member Location: Little Rock AR Join Date: Jul 2010 Posts: 12	Covaris S2 polyA RNA fragmentation conditions This is the only place I have been able to find some starting conditions for RNA fragmentation using the Covaris S2. Using the conditions that Shurjo mentioned (and some longer times, ie upto 3 mins), we are getting 100-500 range centered around 200 bp. What is the ideal range and size for fragmented RNA for Illumina mRNA-seq? Also, Does anyone know if Covaris has come out with specific settings? thanks for your help --K

09-19-2011, 10:55 PM	#16
ETHANol Senior Member Location: Western Australia Join Date: Feb 2010 Posts: 308	I've jsut stated using the Covaris for chromatin fragmentation and so far I'm very impressed with the results. The reproducibility between samples is impressive. What I am wondering is what are the advantages/disavantages of using the Covaris vs. chemical methods for shearing RNA for RNA-seq library preparation? __________________ -------------- Ethan

01-06-2010, 08:26 AM	#2
qdxjcn Junior Member Location: Georgia Join Date: Dec 2009 Posts: 1	Co ask. Did anyone has the experience for RNA shearing to target size fragment with AFA?

01-08-2010, 10:11 AM	#3
Hamid Senior Member Location: San Diego, CA Join Date: Sep 2009 Posts: 105	Hi Shurjo, We have a few customer who have tried a 2-25 minute time course of the setting below to shear RNA: 10%dc,5i, 1000cpb. A customer generated an average shearing size of 400-500b only after a 2 minute treatment using a concentration of less that 100ng/ul. My suggestion would be to use the setting above and carryout an initial test time course of 2-25 minutes depending on the concentration of the RNA sample you will ultimately shear. Thank you hamid

01-20-2010, 02:46 PM	#4
shurjo Senior Member Location: Rockville, MD Join Date: Jan 2009 Posts: 126	Hi Hamid, Thanks for the info. These days I routinely use: 10% DC, 5 Intensity, 200 cpb and 75 seconds for 150ng of polyA RNA in a 120ul volume of H2O in a 6x16mm tube. This gives a nice tight distribution around 200bp every time. Regards, Shurjo

02-14-2010, 08:10 AM	#5
nextgen Member Location: East Coast Join Date: Feb 2010 Posts: 26	Covaris works great for RNA shearing. You can get a very tight range. Just play around with it.

02-14-2010, 08:13 AM	#6
nextgen Member Location: East Coast Join Date: Feb 2010 Posts: 26	I used 500bp range as this right range for 454 (some mRNA transcripts are in that range.) You can easily get smaller size. But for random or end-to-end cDNa synthesis, 500bp is better.

03-04-2010, 01:27 AM	#7
ik76 Junior Member Location: London, UK Join Date: Jan 2010 Posts: 9	Hello, I was just wandering whether anybody have tried fragmenting total RNA using Covaris ( as a replacement for the fargmentation in zink solution recommnded in the Illumina mRNA sample prep protocol). If so, what was the starting concentration and Covaris conditions? Did you have problems with RNA degrading ? if you have started with purified mRNA, how did you measure the conc ? Will just using Agilent Pico chip work ?

09-15-2011, 11:50 PM	#13
RCJK Senior Member Location: Australia Join Date: May 2009 Posts: 155	I just thought I'd give this thread a bump and see if there are any updates regarding using Covaris for RNA shearing, in particular for 454 libraries? We've recently obtained a Covaris and I'm hoping to try it out instead of using Roche's fragmentation procedure. Thanks!

09-16-2011, 07:42 AM	#14
kshankar Member Location: Little Rock AR Join Date: Jul 2010 Posts: 12	RCJK We use the following conditions for mRNA, 120 μl volume, 10% DC, 5 intensity, 200 cpb, 7.5 min and get highly reproducible 150 bp fragmentation peaks. We are using these for Illumina libraries. If you want longer length just shorten the time. In our hands, 30 sec fragmentation gives ~ 500 bp peak, 60 sec ~ 400 bp, 180 sec ~ 200 bp fragments. hope that helps. -Kartik

09-19-2011, 08:12 PM	#15
RCJK Senior Member Location: Australia Join Date: May 2009 Posts: 155	Thanks Kartik!

02-03-2016, 01:29 PM	#18
Hamid Senior Member Location: San Diego, CA Join Date: Sep 2009 Posts: 105	Physical shearing is better since it is concentration independent, isothermal, easily controllable as compared to chemical methods, and you don't have to deal with the stringent cleanup methods required after chemical/heat fragmentation. Thank you.