For the sake of condensing information, I would like to put together Luis and Ivan contributions.

You'll find useful information on this link from Ambion: http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html

Regarding this thread, the most important points are:

- DNAseI cleaves preferentially dsDNA, but can cleave ssDNA with less efficiency (500 times less). You cDNA will be ssDNA (unless you do second-strand synthesis), so it's subjected to cleavage, and that means that you should inactivate your DNAseI before doing anything with it. However, that said, if you do PCR the first step is at 95C... so you'll denature any DNAseI in the solution very quickly.

- RNA is very sensitive to cleavage in the presence of divalent cations and at high temperature.

- You can use EDTA both to prevent DNAseI activity by chelating the divalent cations that it requires (Mg++ and Ca++), AND you must use EDTA if your inactivation protocol involves heat, to prevent cation-related RNA cleavage. Check this paper for more info http://www.biotechniques.com/multimedia/archive/00011/00292bm11_11562a.pdf

Thanks . The thing is that based on this protocol,RNA is going to degrade at 75 degree?: This is a note from the Bio lab for using EDTA in DNase:

EDTA should be added to a final

concentration of 5 mM to protect RNA from being

degraded during enzyme inactivation

The purpose of the EDTA is to stop the DNAse so that it doesn't destroy your cDNA after it is Rev Transcribed. If your cDNA purification is clean enough to remove all DNAse, then you don't need EDTA, but you are taking a risk. In my experience, most kits are good enough at cleaning up DNA that this should not be a problem, but adding EDTA is super easy, so it's your choice.

Thanks Ivan. I am just wondering by heating RNA up to 75 C, dont we degrade it while trying to treat it with DNase?

Ha, a quick googling finds another RG discussion on this point :)

https://www.researchgate.net/post/RNA_integrity_and_heat_degradation

I did cDNA Synthesis more than 100 time, you can inactivate DNAase at 75°C. I see no reason why RNA has to degrade at 75°C, if you work clean without RNAse contamination. Ensure that you DEPC in the DEPC water is properly deactivated. This is more important.

I have done DNase treatment in my RNA samples many times. I have used DNase from invitrogen and according to their protocol, deactivation should be done at 65 centigrade. I don't see any problems in RNA degradation. If you are worried with 75 temperature then use DNase from invitrogen.

Hi,

The protocol already has an enzyme inactivation step: 75degrees /15mins. I do this all the time myself and trust me RNA does not get degraded. Actually, even 65-70degrees for 10mins is sufficient to inactivate the enzyme. After this, you can simply proceed towards RNA precipitation and purification.

The thing is that, I have these RNA samples in -80 from one year ago and now I want to synthesize cDNA from them but I want to do a DNase treatment first.

Maybe you would like to check first if RNA is intact? If it is ok perform DNAse digestion and purify RNA using

https://norgenbiotek.com/display-product.php?ID=561

http://www.zymoresearch.com/direct-zol

RNeasy MinElute Cleanup Kit - Qiagen

or any other RNA purification kit. I recomend directzol because it includes Tri-reagent treatment.

before going for DNAse treatment you should check is there dna contamination in your RNA sample. if it is there then and then only proceed for DNAse treatment. DNA contamination is simply checked py using your RNA as a template in PCR. if band seen then there is DNA contamination if not then your RNA is DNA FREE.

15 min in 75oC should be enough to terminate DNase.

we could use the 75 degrees. as during the RACE reactions(before cDNA synthesis) we use to put RNA at 85 degrees to remove secondary structure from RNA .

for long term storage of RNA we use -- 80 degrees.

For removing genomic DNA from your RNA prep you can use an easily inactivated (1 min at 65 degrees) dsDNase from shrimp, also having no ssDNA or RNase activity (see http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0010295). Is produced by ArcticZymes (http://arcticzymes.com/technology/double-strand-specific-dnases/).

This protocol addresses the issue of genomic DNA contamination in RNA samples using the Ambion (Applied Biosystems) TURBO DNase. We want to remove DNA contamination to the best of our abilities because our interest in environmental stresses is in the gene products that arise from exposure to a stress condition. Since we convert the RNA back into DNA via reverse transcriptase, and then attempt to amplify target genes that we believe to be responsible for the cell’s stress response, any genomic DNA present could also be amplified, leading us to question whether the final results of RT-PCR are due to the cDNA that we generated, or simply the genomic DNA contaminating our sample. To guard against this, we treat RNA samples with DNase, an enzyme that selectively degrades DNA.

DNAse Treatment

Reaction Components - 50 µL reaction

10 ug of RNA x µL

10 x DNase buffer 5 µL

TURBO DNase 1 µL

nanopure water to bring volume up to 50 µL total

The DNase can only handle reactions with an RNA concentration at most 200 ng/µL, as calculated in the reaction specified above.

1.Remember to add components in this order: water, RNA, buffer, DNase

2.Mix gently and centrifuge briefly

3.Incubate at 37ºC for 20 - 30 min

Your RNA has now been treated with DNase, and you must extract the RNA from the mixture to avoid destroying any cDNA you generate in RT-PCR with DNase that might still be left in the sample.

RNA Extraction

1.You may either proceed in a one step extraction with 1:1 phenol/chloroform solution, or two steps with acid phenol and then chloroform (in all cases, add volumes equal to your RNA sample’s volume).

2.Transfer the aqueous phases to new tubes. Avoid the interface. If you are performing the two step protocol, add the chloroform here, and extract the aqueous layer.

3.Add 3M sodium acetate (pH 5.2) equal to 1/10th the volume of your sample to your RNA sample.

4.Add 2.5 volumes of 100% ethanol.

5.Incubate at -20ºC for 1.5 hours.

6.Spin at max speed in the 4ºC centrifuge for 10 minutes.

7.Decant the 100% ethanol, and wash the nucleic acid pellet with 70% ethanol twice.

8.Dry the pellet and resuspend in nanopure water.

9.Nanodrop the resulting RNA sample to check for contamination. Proceed with RT-PCR.

DNase buffer contains relatively high amount of MgCl2, that might cleave/degrade your RNA especially during the inactivation step (75°C for 10 minutes), hence, EDTA chelates the Mg to protect your RNA.

For the sake of condensing information, I would like to put together Luis and Ivan contributions.

You'll find useful information on this link from Ambion: http://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html

Regarding this thread, the most important points are:

- DNAseI cleaves preferentially dsDNA, but can cleave ssDNA with less efficiency (500 times less). You cDNA will be ssDNA (unless you do second-strand synthesis), so it's subjected to cleavage, and that means that you should inactivate your DNAseI before doing anything with it. However, that said, if you do PCR the first step is at 95C... so you'll denature any DNAseI in the solution very quickly.

- RNA is very sensitive to cleavage in the presence of divalent cations and at high temperature.

- You can use EDTA both to prevent DNAseI activity by chelating the divalent cations that it requires (Mg++ and Ca++), AND you must use EDTA if your inactivation protocol involves heat, to prevent cation-related RNA cleavage. Check this paper for more info http://www.biotechniques.com/multimedia/archive/00011/00292bm11_11562a.pdf

I perform DNAse I treatment for all my RNA samples before proceeding to any further downstream application and I shall strongly recommend to do it. There are column based DNAse I digestion where RNAs are bound to column and on-column DNAse I digestion is performed. Then you do not need any heat-inactivation step.

If you have the RNA sample already at -80, just do DNAse I digestion followed by EDTA and heat inactivation. Adding EDTA also prevent RNA cleave (as written above) and your RNA is free of DNA and good for all downstream application. If you do not add EDTA and perform only heat inactivation of DNAse I, it is fine and it also works well most of the times. it is not a big step or protocol and the more sure and full-proof the stp, its better. For rare and less abundant RNA it is definitely better.

At high temperature, in presence of divalent cation and RNA has a self-cleaving activity due to presence of 2-OH group. For DNAse I cleavage you need absolute presence of divalent cations like Ca++, Mg ++ etc.

I think you may add EDTA to be on the safe side.

Thanks

Sara

The first thing to check for your one-year-old RNAs, is to measure them with nanodrop and monitor the wavelength curve in the software, then run them on agarose gel to see if they are still intact, by seeing the 2 typical RNA bands, then you may go further. One of the most reliable controls for genomic contamination is the minus reverse transcriptase reaction in your cDNA synthesis. If you see any product amplified in this control, it mostly due to the presence of genomic contamination. Another tip to escape DNase treatment is to design primers in the exon-exon boundaries if there is any intron in your genomic sequence of the gene.

I did the DNase treatment with and without the EDTA for inactivation of DNase I, and no significant difference have been come up. You shall decide to exclude the EDTA if the PCR with your cDNA dose not lead to a satisfactory result, which then one reason can be the inhibitory effect of EDTA (chelating the magnesium for tag activity) in your cDNA.

High temperature should be enough to inhibit DNAses. Keeping normal sterilty handling should prevent the entrance of new DNAses. Remember EDTA can act as an inhibitor in many Molecular Biology reactions and perhaps working with some samples can become a bit more complicate. Classical TE buffer for DNA elution (1mM EDTA) can be replaced by 0,1 mM EDTA and even 0,01mM EDTA with no compromise for DNA preservation...

Thank you Luis: I definitely favour, like you, to use not more than o.1 mM EDTA in TE.

The role of EDTA is to stop DNAse activity so it help to protect your samples.

We find that one more issue to worry about when using DNase treatment is

That it degrades all RNA:DNA hybrids. So if your target RNA bind DNA your DNase

Treatment will destroy your target RNA.

One way to check the background amount of DNA contamination after

RNA isolation is to perform cDNA synthesis with and without reverse transcriptase.

Followed by rt-PCR.

No rev transcriptase control will give you background DNA contamination levels.

You can then decide if you need to use DNAse or not.

For most genes moderately to highly expressed there are

Many magnitudes more copies of mRNA than DNA contamination that may be carried over from RNA isolation. We find this at least for QIAGEN kits.

EDTA in the DNase deactivation is important to prevent the RNA degradation due to divalent ion transesterification such as Mg(in many buffer) since this ion will fasten the RNA degradation. This divalent ion degradation become more concern especially when you treat your RNA with high temperature (such is in DNase deactivation) since the RNA will be in unstructured condition which is the most vulnerable state for RNA. The reason that your friend didn't have any problem with no EDTA is that there was enough RNA in the sample to proceed but the degradation was happening. So If you feel you have plenty RNAs sample to survive this you could still process your RNA sample even without EDTA. But if the amount is really important using EDTA is the best choice.

EDTA is added for a reversible enzymatic activity, solely heating is used to have an irreversible enzymatic activity (from the protocol)

In this case, should we use only EDTA or EDTA-Na2 will be fine too?

I have to do the same thing but I didn't find EDTA alone.

EDTA  is absolutely necessary. Once, I forgot to add that to DNased RNA, and I used that RNA for RT-PCR. The PCR products on the gel looks like a ladder~~~Means DNase was still active in reverse transcription and degraded cDNA. 

But, I  really don't know if EDTA-Na2 works exactly the same as EDTA.....waiting for the answers.

during DNase treatment , for inactivation of DNase you should have to add  EDTA  to RNA but instead of  this you can prefer removing of dnase by chloroform-phenol  method.

Depends on the protocol you are doing after DNase treatment. If you are proceeding straight way after DNase treatment for RNA precipitation using TRIZOL or Kit based purification, then it is not required, as DNase-I will be washed away. If you are not purifying your RNA-treated with DNase-I then certainly you have to treat with EDTA/EGTA or other chelating agents if compatible for downstream processes to counter down the divalent in reaction while inactivating DNase-I in reaction. 

RNA will be hydrolyzed during heating with divalent cations (Mg++ as a component of the DNase buffer) in the absence of a chelating agent. If you do not want to add EDTA, perform a phenol/chloroform extraction and nucleic acid precipitation or use any RNA clean-up and concentration columns