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Old 07-26-2017, 04:36 AM   #1
patkrat
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Default Does TURBO DNase treatment cause RNA degradation (see bioanalyzer results attached)?

I extracted total RNA from Drosophila melanogaster heads using TRIzol. After dissolving the tRNA pallet, I took an aliquot for later analysis (=pre-DNase tRNA). I then commenced with TURBO DNase treatment (using the inactivation reagent, i.e. the DNA-free kit, so no heat inactivation), and finally purified the DNase-treated RNA using Qiagen RNeasy columns (=post-DNase tRNA).

I have attached bioanalyzer traces for pre- and post-DNase tRNA. There is a shoulder developing for post-DNase tRNA, and I am unsure whether this is due to degradation of RNA, or maybe due to contaminants (maybe from the DNase buffer..). Does anyone have any insight into this?

I also ran a PCR on those samples, and could confirm that pre-DNase treatment tRNA contains gDNA contamination, which is completely gone (35 cycles) after DNase treatment.
Attached Files
File Type: pdf pre-DNase.pdf (79.6 KB, 26 views)
File Type: pdf post-DNase.pdf (83.1 KB, 25 views)
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Old 07-27-2017, 04:57 AM   #2
pmiguel
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Please don't abbreviate "total RNA" as "tRNA". "tRNA" is "transfer RNA".

Yes, the degradation you do see is likely due to the incubation during which any persisting RNAses in your prep could have at your RNA. Unlikely that a reagent that is sold as "RNAse-free" would be the source of RNAase. Not impossible, of course, but far more likely that the RNAse derives from your RNA.

There is a "catch-22" situation with doing a DNAse step prior to a final RNeasy column. That is, if you did the RNeasy column first, then it would likely remove all persisting RNAses from your prep and protect it from degradation. But then you would need to run a second column after doing the DNAse step.

Qiagen RNA prep kits generally offer a DNAse method as "optional" where you bind your RNA to a column, hopefully removing any persisting RNAses and then do an "on column" DNAse treatment. Then you wash and elute -- avoiding the need for a second column.

That amount of degradation may be small enough that you can still construct a poly A+ library of high quality from it.

--
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Old 07-27-2017, 06:35 AM   #3
Dorota
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I don't have any insights into whether in your particular case what you see is RNA degradation or contaminants from TURBO DNase. Although your 'post' photo is after RNeasy cleanup which in theory purify the RNA from contaminants.

I work with samples containing <1ng of RNA /uL and we use TURBO DNase kit without experiencing degradation of RNA which in my case would mean no RNA anymore :P So my intution would tell me that your RNA after TURBO treatment even if it degrades a bit, it should not be a significant amount.
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Old 08-14-2017, 01:46 AM   #4
haixuejia
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I guess there is something wrong with your RNA ladder( forgot denature?) or marker.
Because total RNA should not perfom the band as in your figure( the 28s and 18s bands are too closed). May be you should check the protocol, even the 2100 hardware.
wish you lucky
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Old 08-14-2017, 03:00 AM   #5
patkrat
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Quote:
Originally Posted by haixuejia View Post
I guess there is something wrong with your RNA ladder( forgot denature?) or marker.
Because total RNA should not perfom the band as in your figure( the 28s and 18s bands are too closed). May be you should check the protocol, even the 2100 hardware.
wish you lucky
Thank you for your input. However, this is insect RNA, hence the close double-peak. This is to be expected from Drosophila total RNA.
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Old 08-14-2017, 08:12 AM   #6
seqtechno1
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Default electropherogram error

Hello,
trying to be of some assistance here, what were your errors? If you look on the simulated gel to the right there is a red dot, which indicates an error has occurred, whether some peak on the ladder could not be resolved, improper volume in one or more wells, etc.
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Old 08-16-2017, 03:25 AM   #7
patkrat
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Quote:
Originally Posted by seqtechno1 View Post
Hello,
trying to be of some assistance here, what were your errors? If you look on the simulated gel to the right there is a red dot, which indicates an error has occurred, whether some peak on the ladder could not be resolved, improper volume in one or more wells, etc.
Hi, and thank you for your response. This is insect RNA (Drosophila), and it is well known in the field that 'errors' will always occur on the Bioanalyzer, due to different compositions of the 18s and 28s rRNAs; This is therefore not a problem at all. What simply worried me was the increased degradation that can be seen.
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Old 08-20-2017, 03:58 PM   #8
RNA_
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Quote:
Originally Posted by patkrat View Post
I have attached bioanalyzer traces for pre- and post-DNase tRNA. There is a shoulder developing for post-DNase tRNA, and I am unsure whether this is due to degradation of RNA, or maybe due to contaminants (maybe from the DNase buffer..). Does anyone have any insight into this?

If contaminants are giving rise to the shoulder you might see evidence of them by nanodrop. Precipitating the RNA and washing 2 - 3x with 75% ethanol usually helps me.
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