[betacamp]

Dear pmiguel...

I sincerely hope that yours humility is greater!
First, to make it clear (if you're talking about my posts?)...

I'm not a graduate student, I am a postdoc.

Second, the figure of my sample on the link below, with the 7.30 RIN... tell me something more concrete?!
I made an RNA-seq with this sample on Illumina and I got awesome result, IN MY CASE without DNase treatment!

http://s1244.photobucket.com/albums/...CMYRNA-SEQ.png

Unbelievable, I thought this place was for healthy discussion, but I'm not seeing that!

To: NormSci
I think we should create a post: Suggesting changes in protocols for Illumina Hi-seq!!!!!!!! (Who's more?)!!!

Or... Make a RIN 10!!!!

[pmiguel]

Hey betacamp,
Don't be so sensitive! I was not referring to anyone in this thread with the "hapless grad student" comment.
But I should mention that DNAse treatment is unlikely to increase your RIN score. That is not the point. It is degrading residual DNA that will contaminate all RNA preps. But because of the incubation step, any RNAses remaining in your RNA prep would tend to degrade your RNA. So my advice is to modify your RNA prep method until you can do a DNAse treatment without degrading your RNA.
If you can't get your RNA to stop degrading at this step -- I guess you are right, you must skip the DNAse treatment. But it may cause problems for you later.

--
Phillip

[NormSci]

I completely agree that that DNAse treatment of RNA should be standard protocol prior to RNA-seq, and I hope I'm not giving the impression that it is not. It certainly would not be wise to skip this step as a matter of convenience.

However, for those same hapless graduate students who have tried everything under the sun, yet their RNA continues to degrade after DNAse treatment, I want to point out that all is not necessarily lost, and that depending on their experimental objectives (i.e., sequencing targets), their RNA-seq experiment may be salvageable.

[Papaveraceae]

Zymo Columns

Phillip,

I'm quite new to RNA work but I have managed to get some pretty good results on my pre-DNased samples, see image. I recently read your comment about using the Zymo spin columns to clean up samples after DNase treatment. I was wondering if any effect on the total RNA profile has been reported after using these columns. I've read that many people tend to avoid the RNA extraction kits due to the deleterious effects on the total RNA profile caused by the columns used in these kits. Also, wouldn’t you need to run your samples on the bioanalyzer or nanodrop prior to the DNase treatment, inorder to determine how many units of DNAse to use on your sample.

[pmiguel]

Phillip,

I'm quite new to RNA work but I have managed to get some pretty good results on my pre-DNased samples, see image. I recently read your comment about using the Zymo spin columns to clean up samples after DNase treatment. I was wondering if any effect on the total RNA profile has been reported after using these columns. I've read that many people tend to avoid the RNA extraction kits due to the deleterious effects on the total RNA profile caused by the columns used in these kits. Also, wouldn’t you need to run your samples on the bioanalyzer or nanodrop prior to the DNase treatment, inorder to determine how many units of DNAse to use on your sample.

I think if your RNA is degrading during the DNAse digestion, then it was not purified sufficiently in the first place. You probably got off easy. If it degraded during DNAse treatment at least you know you need to modify your prep. Otherwise you submit it for sequencing and it degrades during library creation. Plus you end up sequencing DNA as well as RNA.

--
Phillip

[Papaveraceae]

...........................

[Papaveraceae]

I think if your RNA is degrading during the DNAse digestion, then it was not purified sufficiently in the first place. You probably got off easy. If it degraded during DNAse treatment at least you know you need to modify your prep. Otherwise you submit it for sequencing and it degrades during library creation. Plus you end up sequencing DNA as well as RNA.

--
Phillip

Phillip,

I made no mention of my RNA degrading after DNAse treatment, in fact I havent treated them with DNAse at all yet, on the contrary the RIN of 9.3-9.2 from my non-DNAsed RNA (extracted using TRI reagent) listed on the electropherogram that I posted is indicative of high quality RNA. Nor am I a proponent of not using DNAse on RNA samples. My message was in regards to the Zymo colums, I was wondering if they had any ill effects on the RNA profile.

Thanks,

Dave

[pmiguel]

Phillip,

I made no mention of my RNA degrading after DNAse treatment, in fact I havent treated them with DNAse at all yet, on the contrary the RIN of 9.3-9.2 from my non-DNAsed RNA (extracted using TRI reagent) listed on the electropherogram that I posted is indicative of high quality RNA. Nor am I a proponent of not using DNAse on RNA samples. My message was in regards to the Zymo colums, I was wondering if they had any ill effects on the RNA profile.

Thanks,

Dave

Hi Dave,

What do you mean by "RNA profile"? If you mean what the RNA sample looks like on an Agilent nanochip, then yes, various people have complained of decreasing RIN scores (sometimes drastically) after DNAse treatment. If that is what you mean, then I refer you to the answer I gave previously.

If by "RNA profile" you mean the actual composition of your RNA sample before and after DNAseing as, for example, assayed by RNAseq -- well I have no reason to suspect that would change. But any purification has the potential to bias sample composition. It would be pretty hard to assay though and seems far-fetched, so the chances that someone actually did an experiment of this sort seem low.

If you mean something else, then maybe you would be will to invest 10% of the time it takes me to answer your post in composing it -- specifically by defining "RNA profile" as it exists in your idiolect!

No, you do not "have" to run an Agilent chip prior to DNAsing your sample. I mean, unless you have some sort of contractual obligation to Agilent of which I am unaware. You could use some other method of quantitation, if you like. But, I am just stating the obvious here.

--
Phillip

[Papaveraceae]

Hi Dave,

What do you mean by "RNA profile"? If you mean what the RNA sample looks like on an Agilent nanochip, then yes, various people have complained of decreasing RIN scores (sometimes drastically) after DNAse treatment. If that is what you mean, then I refer you to the answer I gave previously.

If by "RNA profile" you mean the actual composition of your RNA sample before and after DNAseing as, for example, assayed by RNAseq -- well I have no reason to suspect that would change. But any purification has the potential to bias sample composition. It would be pretty hard to assay though and seems far-fetched, so the chances that someone actually did an experiment of this sort seem low.

If you mean something else, then maybe you would be will to invest 10% of the time it takes me to answer your post in composing it -- specifically by defining "RNA profile" as it exists in your idiolect!

No, you do not "have" to run an Agilent chip prior to DNAsing your sample. I mean, unless you have some sort of contractual obligation to Agilent of which I am unaware. You could use some other method of quantitation, if you like. But, I am just stating the obvious here.

--
Phillip

Phillip,

Sorry I should have clarified. I was using "RNA profile" as a general term to encompass: RIN scores, concentration, RTPCR, and RNAseq. I’m sure there’s a better term out there. I basically want to know if anyone has compared the effects of DNAse treatment alone vs. DNAse treatment + column purification on the same sample using any of the aforementioned tests. It just seems like using column purification would defeat the purpose of the TRI extraction, which “isolates a whole spectrum of RNA molecules rarely observed in RNA isolated by other methods. Typically, the column-based methods may artificially change the mRNA composition." (http://www.mrcgene.com/tri.htm), obviously there's some capitalistic motives behind this statement, but it still seems better to avoid column purification. There are other methods for removing DNAse, its digestion products, and divalent cations from solution without column purification. Check out TURBO DNA-free™ (http://products.invitrogen.com/ivgn/product/AM1907). As for your derisive comments towards me, I would address them but that would require stooping down to your level.

Cheers,

Dave

[Al Merry]

I have total RNA samples prepared using the Ambion Mirvana kit containing miRNA which I am interested in sequencing, as well as larger RNA species, so I need both small and large RNAs to survive downstream procedures. I now need to DNAse them, hence my reply to this thread. I have done a few reactions using a Qiagen DNAse in solution then purifying on an RNEasy Minelute column, which typically gave me about 65-70% recovery from 1-3ug (which is acceptable, although if anyone routinely gets greater recovery I'd love to hear how you do it). But some samples repeatedly give only about 20% yield, which is obviously unacceptable - perhaps those particular RNA samples contained RNAses, but the RIN and everything was great before the reaction.

I have been advised to use the Ambion Turbo DNAse, which I avoided before because I hate using the inactivation resin - in the past using its predecessor, I used to spin the supernatant twice and leave quite a lot behind to ensure no resin carry over. I've been told that column purification is the way to go. My question is, do you use the Turbo DNAse inactivation resin before column cleanup or do you just put the DNAse reaction straight onto the column?

And I've read about people worrying that DNAse treatment will just chop up the DNA into smaller bits that will add background to miRNA sequencing - is this a valid concern?

Thanks for any help,

Al

[maryluzyl]

help how can i use zymo columns

Hi Phillip
I have a problem with DNA contamination in my RNA samples for RNA seq. So you said you use zymo columns for DNAse remotion, what is the protocol for DNAse remotion, how many steps? Do you elute RNA with DEPC water?
Thanks so much
Mary Luz

Listen, you can attempt to convince yourselves that DNAse treatment is not necessary all you want, but that does change the fact that failing to do it can result in DNA sequence contamination of your RNAseq results.

Also, if your RNA is degrading during DNAse treatment, the simple explanation is that your preps still have RNases in them. That is, your RNA prep has been unsuccessful at completely removing them. So, even if you skip the DNAse step, to spare your RNA, all you accomplish is a circumvention of the QC step -- your RNA will likely degrade during a later step.

A "standard paradigm protocol" would have you isolate intact RNA free of contaminating RNases and depleted of most DNA. Then DNase treat to remove the remaining DNA, followed by some purification to remove the DNase and oligonucleotides. (I like Zymo columns for this purpose.)

No one likes to see their RNA degrade during DNase treatment, but better it happens there than at a later step where library QC may not catch it.

--
Phillip

[Apis37]

I don't see how contamination (a little or a lot) can cause differential expression between genes in a RNA-Seq analysis. The DNA is not present is different numbers between samples, so if there is contamination its just noise. Saying you are going to reject any paper that does not do a DNAase treatment is nonsense. I think the biggest problem right now with genomics is people inventing rules based on what they imagine should be the case, when really these techniques are new and we do know exactly what is and isn't necessary.

[armins]

I recently extracted RNA from 20,000 sorted cells that I am going to perform RNA-seq on. Using RNeasy Micro/Mini kits, I would normally get ~ 50 ng total RNA in 20 µL. Given the minute amounts of RNA I was dealing with, I decided to skip the on-column DNase treatment. My Bioanalyzer results show that with the micro kit I have DNA contamination in my samples. The samples prepared by the mini kit are usually free of DNA contamination - only based on the Bioanalyzer results.

I decided to go ahead with what I have and do the RNA-seq, hoping that the poly(A) selection in the TruSeq kit will get rid of most of the DNA. Plus, I did not really have any other choice (because my total sample is so little). If I were to repeat my samples preps, though, I would stick to RNeasy Mini rather than Micro, as it seemed to yield better results, even though advertised otherwise.

I will evaluate my sequencing reads for the presence of intron/intergenic sequences and post the results on here. Best of luck all -

[pmiguel]

I recently extracted RNA from 20,000 sorted cells that I am going to perform RNA-seq on. Using RNeasy Micro/Mini kits, I would normally get ~ 50 ng total RNA in 20 µL. Given the minute amounts of RNA I was dealing with, I decided to skip the on-column DNase treatment. My Bioanalyzer results show that with the micro kit I have DNA contamination in my samples. The samples prepared by the mini kit are usually free of DNA contamination - only based on the Bioanalyzer results.

I decided to go ahead with what I have and do the RNA-seq, hoping that the poly(A) selection in the TruSeq kit will get rid of most of the DNA. Plus, I did not really have any other choice (because my total sample is so little). If I were to repeat my samples preps, though, I would stick to RNeasy Mini rather than Micro, as it seemed to yield better results, even though advertised otherwise.

I will evaluate my sequencing reads for the presence of intron/intergenic sequences and post the results on here. Best of luck all -

I don't get it. How can a bioanalyzer chip estimate the amount of genomic DNA in your sample? Max size detectable on a bioanalyzer (nano or pico RNA chip) is around 10,000 nt. Genomic DNA is 50,000+ bp.

--
Phillip

[armins]

Phillip,

All I know is that gDNA fragments are present in some of my samples, based on the shape of the "inter-region" (I'm trying to attach one of the Bioanalyzer results). I failed to mention that due to the processing of my samples, gDNA is sheared into pieces, so it would show up on the Bioanalyzer graph.