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Amount of DNase to use (Qiagen) - (Aug/16/2010 )

Hi all, I hope this is an easy question but it had me thinking about it the whole day. Sorry if it is too silly.

Today I checked Qiagen's RNase-Free DNase Set webpage (http://www.qiagen.com/products/accessories/rnase-freednaseset.aspx#Tabs=t2) and found this protocol posted there (http://www.qiagen.com/literature/render.aspx?id=23578). In the protocol it says to add 0.5 kunitz units of DNase I to the DNase digestion mix (a 20ul rxn). On the other hand, if you check the RNase-Free DNase Set "handbook" (http://www.qiagen.com/literature/render.aspx?id=164) the 50rxn set comes with 1500Kunitz units which, divided by the 50rxns for which the kit is marketed yields 30kunitz units per reaction, which is 60! times more DNase than what the protocol says to add per reaction!.

My question is: are my calculations right or I am missing something? alternatively does anybody knows whether Qiagen's protocol contains a typo or if it is reliable? I need to do lots of RNA extraction for qRT-PCR in the future and I was wondering whether I could be more efficient in using the DNase. I know the kit is "relatively" cheap compared with other reagents, but if my calculations and the posted protocol are right, I could make the kit 60 times cheaper of what it is now.

Thank you very much in advance.

-echica-

echica on Tue Aug 17 00:50:21 2010 said:


Hi all, I hope this is an easy question but it had me thinking about it the whole day. Sorry if it is too silly.

Today I checked Qiagen's RNase-Free DNase Set webpage (http://www.qiagen.com/products/accessories/rnase-freednaseset.aspx#Tabs=t2) and found this protocol posted there (http://www.qiagen.com/literature/render.aspx?id=23578). In the protocol it says to add 0.5 kunitz units of DNase I to the DNase digestion mix (a 20ul rxn). On the other hand, if you check the RNase-Free DNase Set "handbook" (http://www.qiagen.com/literature/render.aspx?id=164) the 50rxn set comes with 1500Kunitz units which, divided by the 50rxns for which the kit is marketed yields 30kunitz units per reaction, which is 60! times more DNase than what the protocol says to add per reaction!.

My question is: are my calculations right or I am missing something? alternatively does anybody knows whether Qiagen's protocol contains a typo or if it is reliable? I need to do lots of RNA extraction for qRT-PCR in the future and I was wondering whether I could be more efficient in using the DNase. I know the kit is "relatively" cheap compared with other reagents, but if my calculations and the posted protocol are right, I could make the kit 60 times cheaper of what it is now.

Thank you very much in advance.


I'm not familiar with Kuntiz units, but it says there are 2500 Kunitz units/mg. But they say there are 1500 units of DNase. I don't think Kunitz units are the same as units (U). Umm, hopefully that made some sense. I don't think it's a typo.

Our lab uses DNase I from Ambion--it comes also as 1500 units. We basically just have to add a special 10x buffer, then the RNA, then we do a sequential 0.5 ul DNase I incubation at 37 degrees for 30 minutes followed by 0.5 ul more of DNAse I at 37 degrees for 30 minutes.

-b

-btc8-

Thanks btc8 for your reply, I am still a bit confused. I checked Ambion's DNase specification sheet (http://www.ambion.com/techlib/spec/sp_2222.pdf) and it says that one Unit (U) is equivalent to 0.04 Kunitz units, so as you pointed, Kunitz units are not the same as Units (U). Thus, I still believe there is a typo somewhere in Qiagen's literature because in the first page of the "handbook" it says the kit comes with 1500 Kunitz units and in the second page it says the kit comes with 1500 Units.

This makes me think that there is also a typo in Qiagen's protocol maybe they mean to use 0.5 Units (not Kunitz units) as this would be more in line with what other providers of DNase suggest (that would be about 12 Kunitz units per reaction).

I will try to contact Qiagen directly and and see what they say.

Thanks again!

-echica-

Did you try without using DNase digestion?
I'm not sure if the difference is significant between using DNase digestion or not using it! I have carried out many qPCR without DNase digestion and input wa OK.

-Biog-

Regarding Kunitz: the specific activity of a DNase I preparation reflects the potency of the enzyme per unit mass in degrading double-stranded DNA (dsDNA). Historically, this activity has been expressed in Kunitz, where 1 Kunitz is the amount of DNase I added to "highly polymerized DNA" that causes an increase of 0.001 A260 absorbance units per minute per ml when assayed in a 0.1 M NaOAc (pH 5.0) buffer at 25C.

The problem here is that these conditions do not represent those used for typical for DNase I digestions (0.1 M NaOAc, 25C). Moreover, there's no way to know the "degree of polymerization" -- whatever that is -- of your target DNA (most manufacturers use a standard DNA template when assaying for Kunitz, e.g. 1 mg/ml salmon sperm or lambda DNA). Additionally, done exactly as stated with absolutely pure reagents, I don't see how any DNase I activity would be detected under the Kunitz assay, as there is no calcium or magnesium present. So, to my mind, the Kunitz measure is fairly useless; at best, it would indicate an activity level far below that which would likely occur under typical usage conditions.

Consequently, most manufacturers provide their own unit (U) definition, where the enzyme's potency has been assayed under conditions more closely resembling the conditions that are likely to be used, or under optimal conditions (e.g. "1 unit of DNase I is defined as the amount of enzyme that will degrade 1 g of DNA in 10 min at 37C in 1X DNase I buffer <10 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2>"). If there is such a unit (U) definition, I would use that rather than Kunitz.

-HomeBrew-

Thanks Biog and HomeBrew for your replies.

@ Biog - I have tried with and without DNase digestion a couple times in the past and agree I have never seen any difference between DNased and non-DNased samples in my trials. I do phenol-chloroform precipitation followed by column purification and I guess I should have very little (negligible) amount of contaminant DNA. Nonetheless, my advisors would not allow me to go without DNasing my RNA for the sake of keeping good laboratory practices, at first I did not agree too much with this, but now I willfully adhere to them, since you never can be too sure. As I said, the times I have tried to test this I have not have any problem, but I know people who has had significant DNA contamination. So I guess I will keep it in my protocol.

@ HomeBrew - I did not know all those details about Kunitz units, but it sounds pretty useless. It is still shocking to me what I believe is atypo in their handbook since in one place the say "1500 Kunitz units" and in another they say "1500 units" and that their protocol does not seem to agree with how they market the kit.

I emailed tech support last week when I posted this topic and I have not received any reply until today. I emailed them again this morning I will post here what they say.

On the other hand, I was thinking about the protocol issue and thought perhaps they market the kit for on-column digestion and the protocol they offer is for in-solution digestion. I can think of a couple of reasons why to use different concentration of enzymes if done in different media. But again this is just a thought I had, I will wait for the official answer from the manufacturer. If you have any other thought, it would be greatly appreciated.

-echica-

Hi again, I got a response from Qiagen. Page 2 in the handbook should read Kunitz units instead of just units, and about the protocol below I pasted my question and the answer I received:

Q: Further, in the protocol that accompanies the RNase-Free DNase Set (http://www.qiagen.com/literature/render.aspx?id=23578), it indicates to use 0.5 Kunitz units of DNase I per reaction. This means that each RNase-Free DNase Set which is marketed for 50rxns (1500 Kunitz units) could actually be used for 3000 reactions (!?). Surely I am overlooking something, please could you indicate where is my mistake?


A: This protocol was established some time back a a beanch guide and is for use with other suppliers of Dnase I. Of course, you can use our DNase I but again at 0.5 Kurtiz Units.

In all our kit developed protocols we recommend the use of the Rnase-Free Dnase I set (Cat # 79254) that comes in a 1500 Kunitz Units. This DNase I comes lyophilized and is resuspended in 550ul of Rnase-free water. For each Mini Prep you need 10ul of this stock Dnase I for On-column DNA digestion (~27K Units) and 2.5ul for Of-column DNA digestion ( 6.8 Kunitz Units) in the presence of our RDD buffer.


I am not satisfied, but at least they answered, I thinking on determining the amount of DNase to use for my extractions empirically.

Thanks all for your comments!

-echica-

Hi all,

I'm new to the forum but need a reply asap on this:

Can I use DNAse (ambion) to treat my RNA sample in Qiagen column?
I have used before qiagen DNAse with RNAeasy kit...worked fine but now I only have the DNAse from ambion which my colleagues use for off column RNA tratment.

Has anyone tried that before? what protocol did you use? I'm thinking to use 1 uL DNAse + 5 buffer and 40 uL DEPC water to trat my column...Would this be fine?

Thanks

-zepedrof-