Juliane, I am not sure I understand your question. But from what I gather, you are saying that you are treating you cells with a substance that interferes with cDNA synthesis and PCR. I sort of fail to understand how this is the case? Are you washing your cells 3-4 times with PBS before RNA isolation or are you talking about getting RT- contaminations? If it's RT- contamination, how about using new reagents from RNA isolation to cDNA synthesis steps?

You can also run your RNA on a gel to determine any genomic DNA contamination.

Which kits do you use for RNA isolation?

you could add constant amounts of exogenous RNAs such as luciferase RNAs or such like to your reverse transcription reactions. in the subsequent qPCR you can quantify whether your substance affects reverse transcription.

You should be more specific, I can't imaging what kind of substance are you talking about. A basic step is the RNA isolation previous to RNA transcription, probably using a RNA isolation kit with poliT magnetic beads you would not carry over any substance (unless this substance interacts with the RNA somehow). Another try could be to perform the RT with poli T primer instead of with random hexanucleotids maybe it would work better in your system. However you would have to perform the PCR to check the result. As far as I know, you have to perform the PCR after the RT to know if it has worked properly. You could check if that substance affects the PCR, use it in a PCR with other primers and template and add to the PCR mix that substance at the same concentration than when you are isolating the mRNA, besides run the same PCR without the substance and compare the result. With this experiment unless you could know if the TaqPol is affected or not by your substance. Another posibility, you could measure the yield of cDNA produced using a nanodrop.

You want check directly the Reverse Transcription, however can be hard stablish if this reaction works without a subsequent PCR. Try to check your RT amplifying a constitutive expression gen, if this doesn't works, is very probable that your RT can be inhibited. Include RNA without treatment like positive control!

Is it possible to check whether your "substance" interferes with DNA amplification instead? As suggested by others, you can also wash your cells extensively to get rid of the interfering material and also use bead methods to isolate RNA.

If the substance enters the cell, which it probably will, extensive washing might not be of much help

Hello Juliane, I never had this kind of problem, but I read a paper whose the authors speak about this, and she purpouse a simple test.

The name of the paper is:

SPUD: A quantitative PCR assay for the detection of inhibitors in

nucleic acid preparations, by Tania Nolan a, Rebecca E. Hands b, William Ogunkolade b, Stephen A. Bustin.

Sorry if I don't have a tip, but I hope this paper can be helpful. Good luck!

I agree with Rosa that this does not seem a very plausible scenario. But you could set up a few duplicate PCR reactions with template cDNAs (or any templates) that you know work well, but spike one of each pair with a small amount of your RNA that contains substance X. This amount of RNA should correspond to the amount that would ordinarily carryover into your qPCR step. The "control" would be spiked with a similar amount of RNA from a non-inhibitory prep. If the reaction containing the spiked RNA fails, then you would know that substance X does, in fact, inhibit your PCR reaction. Note that you are spiking with RNA, NOT cDNA, so the added RNA would not itself be a template for any PCR.

I think that the first step to check is you RNA by capilar eletrophoresis that will give if you RNA is degraded enought to make your RT or not. the agilent system bioanalyser 2100 with the RNA specific device is very easy to se by RIN, that could be higerh than 9.0 , look carefully the electrophoresis gel to see if you have large amount of small peak, if so your RNA is degraded. nMust appear only 2 peak charply.

if the RNA is not good, just see the how you conseved your samples, to avoid degradation, using RNA later or similar product.

so if your kit to extract RNA is ok, just see by housekeeping gene. amplification.

you can run a RT product in the same eletrophoresis as your RNA to see if double strand DNA formed. (change RF.

good look

mario

Thank you for the many answers up till now. I'm working with Glycosaminoglycans (polysaccharides). The problem is that real-time PCR with samples from cells treated with high sulfated GAGs doesn't work. No Amplification of any genes, not even beta-Actin, GAPDH or other housekeeping genes. For a long time i thought that i made a mistake somewhere, but when i mixed cDNA from a sample of untreated cells that worked fine so far with cDNA from a high dosage GAG sample there was no amplification as well. I isolate RNA with the RNAEasy Kit from Roche(+DNAase treatment) or by Phenol/chloroform. Each methods works fine in respect to the yield but there is a small contamination by salts which i think comes from the GAGs. They accumulate in the cells by the way as someone already pointed out. Any idea how i can get rid of the RNA contamination or how to pimp my PCR ? (i tried other buffers with more mg2+or additives like tween and dmso already). Right now i tried the RNA-spiking and i will measure the resulting cDNA at the NanoDrop. Thanks for this idea!

I already spiked cDNA with GAGs or RNA preps from GAG treated cells. -> Inhibition for the sulfated GAGs. Anyone an idea HOW they interfere?

Hi Juliane, based and the comment I assume that you run your RNA and is for sure ok and clean (at the level that a gel can tell us). Thus, if the problem is the GAGs interferig in your PCR, it could be or during the RT (Have you check if you really have cDNA?) and for sure at qPCR since your mixing tests. If your cDNA synthesis is OK I think your could try to clean your cDNA using an DNA extraction cleaning kit, that should be able to remove the most of non-DNA compounds present in your samples, how ever I think is better to try to clean during RNA extraction, you could try with TRIZOL (or equivalent) + cleaning with columns or a double cleaning. May be it could help to elute and/or dilute your RNA in pure water (doble distiled, RNAse free one) instead of depc treated or TE or other elution buffer, sometimes they make interferences with the PCR reaction, the question is that you should be very careful with keeping your RNA samples all the time in ice while manipulating and frozen fast, since they are not protected by deoc or salts. It could be also interesting to run the results from your qPCR in a gel so can be sure that it is not really working, may be the interference is in the reporting staining and not in the amplification...

It looks like GAGs are potent inhibitors of reverse transcriptases AND DNA polymerases:

http://www.ncbi.nlm.nih.gov/pubmed/61158 (So I was wrong about the possibility of this scenario!)

So you are probably going to have to find a way to specifically purify them away. Have you tried consulting the "RNA experts" at Ambion (now a division of Life Technologies)? Perhaps a size fractionation by gel filtration? There are spin columns containing gel filtration matrix which would be relatively easy and cheap.

http://www.biocompare.com/Protein-Biochemistry/22804-Size-Exclusion-Spin-Columns/

You'd have to pick the matrix which would trap GAG-size molecules (how big are they?), while allowing mRNA size molecules to pass through.

It is good to know!

Oh wow. How could i miss this paper? Funny thing is in yesterdays reverse transcription i got the same amount of DNA in the samples spiked with GAGs and without them. I contacted Life technologies as you suggested :).

Molecular weight of my GAGs ranges from 10^4 g/mol to 10^6 g/mol. (hyaluronan).

How about an enzymatic digestion of the GAGs during the RNA prep with e.g. chondroitinases? The columns we use also have an DNAse treatment step. I would add the enzyme at this step.Or do you think this would make everything worse?

Re: "Funny thing is in yesterdays reverse transcription i got the same amount of DNA in the samples spiked with GAGs and without them."

Nanodrop is not useful for this measurement, since template RNA and, perhaps most importantly, the substrate deoxynucleotides will cause most of the detectable UV absorbance in an unpurified reverse transcription reaction.

Re:"Molecular weight of my GAGs ranges from 10^4 g/mol to 10^6 g/mol. (hyaluronan)" The largest of these are probably not easily/ quantitatively separable from mRNA by size exclusion.

Enzymatic degradation sounds good, but my experience with "on-column" DNAse is that it is nowhere near as effective as enzymatic treatment in solution--presumably because the nucleic acid bound to the column is not as accessible to the enzyme and the buffer conditions are not ideal. Presumably the GAGs are also bound to the column--which is why they come through the purification-- and so they may not be readily accessible to enzymes either. And the degradation products might still be inhibitory..... So if you try that route, I would suggest setting up the reaction in solution with part of your RNA yield, followed by a clean-up that will separate the mRNA from the enzymatic products. After enzymatic degradation, the GAG fragments might be eliminated after a simple ethanol precipitation or trizol extraction followed by ethanol precipitation. It might even be possible to perform the enzymatic GAG degradation on a crude cell lysate prior to the initial RNA extraction--but that might be too likely to result in degradation of your RNA as well.

Did the Ambion people have any useful suggestions?

No surprise then that the values were so similar :(. Damn. Now i transcribed a lot of (spiked) RNA in cDNA in vain :(. Think i will see something when i put the outcome in an ethidiumbromid gel?

I am still waiting for the reply of the ambion people.

I will try the degradation next.

Hm. I just remembered that RNA preps contaminated with DNA had the DNA stuck in the pockets of the gel (1% Agarose). Will this happen for the cDNA generated by Random primers also? And can i discriminate the RNA from the cDNA at all?

The size distribution of cDNA should be generally smaller than the size distribution of your RNA--and at most, the amount of total high molecular weight nucleic acid would double-so an EtBr gel may not show much change even for a successful reverse transcription. It's possible that fluorescence might increase substantially if there is higher binding of EtBr to RNA:DNA hybrids than to RNA tho.....

Two things to keep in mind when you enzymatically treat your RNA. First, you should consider adding RNAse inhibitor to the reactions. We use recombinant RNasin from Promega at 1unit/ul in DNAse reactions (manufacturer recommendation). How important this is depends on how much RNAse was present in your extracted samples. Also, very important to never heat your RNA in the presence of Mg++ ions--this causes substantial RNA degradation. Don't know what the ideal buffer conditions are for your chondroitinases, but if they contain Mg++, don't try to kill the enzyme by heating. Incubation at 37C is ok, but should not be more than an hour or so.

Thanks for all the answers so far. I tried the enzymatic treatment, but it made it even worse. I'll try next on the suggestions of the lifetech-support team. Here is the answer i got from them:

"From how I interprite your query there are essentially two parts: 1) purification of already isolated, GAG contaminated RNA, and 2) Isolation of RNA, without GAG carryover. One additional point I would like to address before I go any further. This is just a word of caution regarding the measurement of your cDNA using the nanodrop. Please note that under normal measurement procedures the nanodrop really struggles to accurately distiguish between DNA and RNA. The use of DNA and RNA specific binding dyes is the only way to be completely sure of this distinction for quantification of these sample types. Regarding your main query. This is an interesting problem that you have. As a possible solution to part 2 above, I would suggest trying our Plant Isolation Aid (AM9690). This is a polyvinylpyrrolidone (PVP) based solution that bind to contaminates found in plant samples, such as polyphenolics and polysaccharides. The reagent is compatible with most isolation procedures that use chaotropic salts for lysis. You simply add the solution to the normal lysis bufer and homogenise your sample within that mixture. As another possible solution to part 2 and also for cleaning up already isolated RNA (part 1), would be reprecipitation/isolation of the RNA with trizol. However, if a sample is known to have a high content of proteoglycans and/or polysaccharides (such as rat liver, rat aorta, plants, your samples), the following modification of the RNA precipitation step should remove these contaminating compounds from the isolated RNA: · Add 0.25 ml of isopropanol to the aqueous phase followed by 0.25 ml of a high salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl (no pH adjustment necessary)) per 1 ml of TRIzol used for homogenization. Mix the resulting solution, centrifuge, and proceed with isolation as described in the protocol. This modified precipitation effectively precipitates RNA and maintains proteoglycans and polysaccharides in a soluble form. To isolate pure RNA from plant material containing a very high level of polysaccharides, the modified precipitation should be combined with an additional centrifugation of the initial homogenate. In general, it is not recommended to use the high-salt precipitation if polysaccharide or proteoglycan contamination is not a concern since it is an extra step and there is no significant advantage to be gained from it. When purifying an RNA sample where polysaccharide/proteoglycan contamintation is not an issue, in general the total RNA yield will be same with or without the highsalt. There may be small changes in the RNA profile reflected by slightly decreased tRNA. The high salt precipitation reduces tRNA in the sample. I hope this information is of help to you Please let me know if you have any additional questions"

Good Luck figuring out what that mass of prose means. Hopefully there was some reference to the specific protocol that is to be modified. Also, for the record, there is no such thing as an RNA-specific fluorescent dye. There are dyes that you can use for fluorescent measurement of pure RNA. But the key word is pure. If you have a mixture of RNA and DNA, the dye will also bind to the DNA.