Welcome to part 2 of our Epigenetics series. In Part 1, we gave an overview about what epigenetics is and the various types of epigenetic modifications that may occur. In this post, we will look at methods used to study DNA and RNA methylation.
DNA and RNA Methylation
DNA methylation is one of the most intensively studied epigenetic modifications, both within research and in vitro diagnostics, especially for cancer where widespread DNA methylation changes often occur early during tumorigenesis. Such methylations are detectable in liquid biopsies as an early screening tool for certain cancers. A number of cancer diagnostic assays based on detection of methylated DNA are already in clinical use.
RNA methylation is a growing area of interest, and many of the identified RNA marks are identical to their DNA counterparts, owing to the similar structure of the DNA and RNA backbones. Both nucleic acids also share the same SAM (S-adenosyl methionine) as the methyl donor, and the methylations are catalysed by methyltransferases.
5-mC and m6A are the most abundant RNA marks and research supports a role for both of these in the regulation of RNA stability and mRNA translation. While the function of RNA methylation is not fully understood yet, recent data implicates abnormal RNA methylation in a number of human diseases, including certain cancers and autoimmune diseases. m6A’s role in mRNA regulation is supported by its enrichment at 3’UTRs. Loss of 5-mC in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Other RNA marks include N-7 methylguanosine and N-1 methyladenosine.
Studying Nucleic Acid Methylation
DNA methylation can be studied in a number of ways, including bisulfite methods, non-bisulfite methods and antibody-based methods, e.g., enzyme-linked immunosorbent assay (ELISA). Often, several methods are used in tandem, and the method(s) chosen depend on the desired outcome, for instance whether the researcher wants to obtain information about methylation at specific loci, or whether an overall picture of the extent of methylation throughout a genome is sufficient.
RNA methylation is most often studied using bisulfite methods, although ELISA kits also exist for global m6A and 5-hmC detection in RNA.
Non-bisulfite and antibody-based approaches will be discussed later in the series, but for now let’s take a closer look at bisulfite-based analysis of nucleic acid methylation.
What is Bisulfite Conversion?
Bisulfite treatment or conversion is considered to be the “gold standard” for the analysis of DNA methylation, and is viewed as the most convenient and efficient way to map DNA and RNA methylation to specific nucleotides.
The starting point is isolated purified RNA-free DNA or DNA-free RNA, which is then subjected to a three-step chemical reaction that takes place between the cytosines present in the nucleic acid sample and sodium bisulfite, which is supplied in the reaction.
Bisulfite conversion results in the conversion of unmethylated cytosines into uracil. Figure 1 illustrates the overall chemical steps involved in a typical bisulfite conversion reaction.
Figure 1. The chemical steps in a typical bisulfite conversion reaction. Unmethylated cytosines are converted into uracil. Typical DNA inputs include purified genomic DNA, endonuclease-digested DNA and linearised plasmid DNA. Typical RNA inputs include RNA and mRNA. Image courtesy of Zymo Research.
The beauty of the bisulfite approach is that only unmodified, i.e., unmethylated cytosines can be converted to uracil while methylated cytosines are protected from conversion. The resulting DNA or RNA can then be analysed by PCR followed by sequencing, hybridisation, methyl-specific PCR (MSP), pyrosequencing or next-generation sequencing to map the positions of methylated cytosines in the original sample (Figure 2).
Figure 2: Example of DNA sequencing chromatogram following bisulfite treatment. The original sample was subjected to bisulfite conversion with the EZ DNA Methylation™ Kit (Zymo Research). The resulting DNA was PCR-amplified and then sequenced. The methylated cytosine at position 5 remains as a cytosine while unmethylated cytosines at positions 7, 9, 14 and 15 have been converted to uracil. Note that these are detected as a thymine following PCR. Image courtesy of Zymo Research.
Handling Bisulfite-Treated Samples
The chemical conversion process is rather harsh and drastically changes the physiochemical properties of the nucleic acid sample. Nucleic acid samples after bisulfite treatment exist as a mixture of randomly fragmented single-stranded molecules. These changes may affect overall nucleic acid quality and recovery and must be considered in the context of downstream analyses and what they may require. Therefore, a thorough quality assessment of treated DNA/RNA is highly recommended before moving forward with further analysis.
Quality can be assessed using electrophoresis, spectrophotometry or fluorometric methods as described and compared in our previous article about RNA quality (most of which also applies to DNA). However, some adjustments should be made as follows:
• If using a spectrophotometer to quantify DNA, a value of 40 µg/mL should be entered for Ab260nm =1 since bisulphite-treated DNA resembles RNA.
• If using agarose gel electrophoresis to assess DNA, a high gel strength, e.g., 2 % and a low molecular weight marker is recommended to resolve the small DNA fragments.
• You may also need to cool your gel in an ice bath in order to see the DNA fragments. This will encourage base pairing between the single-stranded fragments which will in turn allow intercalation by ethidium bromide or other double-stranded DNA-binding stain.
Downstream Analysis: PCR-Based Methods
There are many ways to analyse bisulfite-treated nucleic acids, but PCR-based approaches are by far the most popular. These include bisulfite PCR, methylation-specific PCR (MSP), and bisulfite sequencing or bisulfite pyrosequencing, both of which require PCR amplification of bisulfite-converted DNA. Bisulfite-treated RNA can be subjected to the same methods, once it is reverse-transcribed to complementary cDNA, which then serves as the PCR template.
Bisulfite PCR involves the use of regular primers that can bind and amplify a region independently of methylation status. This type of PCR is generally used to generate material for another downstream method such as mass spectrometry, bisulfite sequencing or bisulfite pyrosequencing that can identify where in the genome the methylated cytosines were located.
MSP on the other hand is used to assess the methylation status at specific loci, and relies on the differential amplification of the template using methylated and non-methylated primer sets.
PCR amplification of bisulfite-treated nucleic acids is inherently error-prone owing to the physiochemical state of the sample following conversion. Success relies heavily on well-designed and tested primers, optimised annealing conditions and a suitable polymerase, e.g., a hot-start polymerase, which will reduce non-specific primer binding.
Bear in Mind
As elegant as bisulfite conversion is, there are a few downsides to this approach:
• Bisulfite conversion does not allow discrimination between various methylation marks, for instance, neither 5-mC nor 5-hmC will be converted to uracil.
• Because bisulfite conversion is a harsh treatment, the input material needs to be of high quality, otherwise the inevitable nucleic acid fragmentation that already impacts sample recovery will lead to significant sample loss = information lost.
• Bisulfite conversion is not a standalone procedure. It always requires follow up with a second step (e.g. PCR, NGS). This makes the workflow long and prone to errors, and choosing a downstream step might not be straightforward. For instance, when PCR is used to analyse bisulfite-treated samples, primer design is crucial to reliable results, and NGS may be costly. For global detection of 5-mC, 5hmC and/or m6-A, antibody- based methods present a quick and efficient solution. These will be discussed later in the series.
Choosing the Right Setup
Choosing the right approach to study DNA methylation, even when committed to the bisulfite strategy may depend on factors such as level of sensitivity required, downstream analysis, available input material and level of throughput needed.
There are many commercial bisulfite conversion kits to choose from, which differ in the rate of bisulfite conversion, format (i.e. tube-, microwell-, or magnetic bead-based) as well as workflow and assay time. Some of these kits offer direct conversion from blood samples, while others streamline the entire conversion reaction with a single buffer in a single step for convenience. Feel free to contact us if you would like to discuss finding the right bisulfite approach for your project.
Related Blog Posts and Useful Resources
Resource: Bisulfite Beginner Guide – Zymo Research. This contains useful information about primer design for bisulfite and methylation-specific PCR.