Use the right controls for RNA modification antibodies

RNA modification antibodies

When you are working with RNA modifications such as m6A and ac4C, it is essential to ensure that your antibodies are specific – only binding to the correct modification. Due to the nature of RNA modifications, their chemical structures are often very similar. To ensure you are getting the most accurate results from your antibodies, you need to test them in your model system thoroughly. Controls for RNA modification antibodies can be done using a range of applications. See below for some of our advanced controls and tips to make your RNA modification research easy. 

• RNase treatment
• DNase treatment
• Competition assays
• Dot blot
• RNA immunoprecipitation-mass  spectrometry (RIP-MS)
• Use a well-established antibody

RNase treatment

Whether you are carrying out ICC/IHC or dot blot, it is essential to have an RNAse-treated control alongside your experimental samples¹ to ensure that you are not picking up non-specific background signal from DNA when using RNA modification antibodies. For example, if you see a clear, bright signal in your experimental IHC samples, but you get no signal in your RNAse-treated control samples, you can be confident that the signal you are getting is from the RNA and not the background signal from a non-specific source. This suggests that the antibody is recognizing the modification within RNA and not the DNA.  

You can quickly add an RNase treatment step to your normal RNA modification IHC, RIP, or dot blot protocol¹. It is important not to leave the samples in the RNase solution for too long, as this can lead to the degradation of the DNA, making it difficult to carry out counterstains such as DAPI. For each different sample type, you should test different RNase concentrations and try leaving on your samples for varying lengths of time. For example, an IHC may need an RNAse time of up to an hour depending on the tissue type, whereas an ICC will require much less time – try 10–30 mins as a starting point. 

DNase treatment

In addition to RNase-treated controls, you should carry out DNAse-treated controls. If you are concerned that your RNA modification antibody recognizes a similar modification within DNA, the best way to test for this is to treat your samples with DNAse. Many modifications are within both RNA and DNA, so this is a common problem. For example, 5mC within DNA has the same chemical modification as m5C within RNA. 
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If you carry out IHC using an RNA modification antibody, it is a good idea to have a DNAse-treated control alongside your experimental samples. If you get a clear, strong signal from your experimental samples, but your DNAse-treated control has no signal, it suggests that your antibody is binding to a modification within DNA. For this type of control, it is also important to optimize the conditions. Leaving your samples in DNAse treatment for too long can lead to degradation of RNA, so be sure to test different DNAse concentrations and the duration of the treatment. 

Competition assays

Another way to ensure the specificity of your RNA modification antibody is to use a competition assay. This assay uses a synthetic modification-containing oligonucleotide, which can be pre-incubated with your antibody². When you then use this pre-incubated antibody for your applications, eg, ICC/IHC or dot blot, you should see a reduction in the signal obtained compared to a sample stained with the antibody alone. You can try adding the competitor oligonucleotide to your antibody solution at increasing concentrations. You will expect to see a decreasing gradient of the signal reflecting the amount of competitor you add to the antibody. For example, try a gradient of 0 ng, 10 ng, 100 ng, and 1µg of the competitor oligonucleotide².

Dot blot

Carrying out a dot blot using RNA modification antibodies can be a quick and simple way to test for their specificity. A dot blot works like a simplified version of a western blot. For this technique, the sample is spotted directly onto the membrane, cross-linked, and then undergoes blotting. For more details, take a look at our dot blot protocol here. If you have access to synthetic RNA molecules containing your modification of interest, this can act as the perfect positive control². Similarly, loading an unmodified molecule or a molecule containing a different modification can serve as a negative control and help you to gauge any non-specific binding or cross-reactivity². 

It is possible for your experimental samples to test whether your RNA modification antibody is specific by carrying out a dot blot with the right controls. For negative control, use samples that contain a knockout (KO) for the enzyme responsible for producing your specific RNA modification, eg, ALKBH5 for m6A^3,4. If you load RNA from your wild-type and KO samples onto a membrane for dot blot, you should see a clear difference between the two samples. The wild-type sample will display a clear signal, and the KO should appear blank when the membrane is stained using an antibody against your RNA modification of interest.  

RIP-MS

If you have access to liquid chromatography-tandem mass spectrometry (LC-MS/MS), then this is really the best way to test for RNA modification antibody specificity⁵. Using this technique combined with RIP (RIP-MS) will allow you to determine if your antibody is binding to your modification of interest, and it will also let you to see if it binds any other non-specific modifications. For more information on RIP, take a look at our RIP protocol here. Or take a look at our miCLIP protocol, optimized for use with our m6A antibody⁶. 

Using either absolute or relative quantification methods, LC-MS/MS gives you parallel quantification of all the RNA modifications found in total RNA from any organism and cell type. If you generate LC-MS/MS data of your RIP input and pull-down samples, you should see an enrichment of your modification of interest in the pulldown sample compared to the input. You can also then check other modifications with these same data to see if anything else came out as enriched in your samples to test for non-specific antibody binding. There is software being developed now that can even help you with this type of analysis⁷.  

Use a well-established antibody

The distribution of abundant RNA modifications such as m6A is well characterized in many model systems. If the modification you are interested in is less well known, you could use a modification such as m6A as a positive control for many applications. For example, if you are carrying out RIP-qPCR and you want to be sure that technically the experiment has worked, you could carry out RIP-qPCR for m6A alongside your experimental samples and choose regions of the transcriptome previously shown to contain m6A as positive control regions. This will help ensure that your buffers, other reagents, and method are all working fine before you try a novel modification. 

References

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2) Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR.(2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 1635-46

3) Jia G, Fu Y, Zhao X, Dai Q, Zheng G, et al. 2011. N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7:885–87

4) Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, et al. 2013. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49:18–29

5) Kellner S, Ochel A, Thüring A, Spenkuch F, Neumann J, Sharma S, Entian KD, Schneider D, and Helm M. (2014) Absolute and relative quantification of RNA modifications via biosynthetic isotopomers. Nucleic Acids Res. 42(18): e142. 

6) Linder B, Grozhik AV, Olarerin-George AO, Meydan C, Mason CE, Jaffrey SR. (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.Nat Methods 12:767-72

7) Yu N, Lobue PA, Cao X, Limbach PA. (2017) RNAModMapper: RNA Modification Mapping Software for Analysis of Liquid Chromatography-Tandem Mass Spectrometry Data. Anal Chem 10744-10752