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In mammalian cells, DNA methylation at the 5-position of cytosine leads to recruitment of proteins that selectively recognize and bind 5-methylcytosine (5mC). Taking advantage of the structural identity of 5mC, various affinity purification-based protocols have been developed to enrich for either DNA that is modified by 5mC or proteins that recognize 5mC. In this protocol, an antibody against 5mC is used to immunoprecipitate the methylated DNA. The method can be scaled up to perform genome-wide DNA methylation analysis. Because immunoprecipitation is a straightforward procedure that does not require any prior modification of genomic DNA, we also describe several commercial kits available to perform the immunoprecipitation-based detection of DNA methylation.

Chemical sequencing of RNA relies on the fact that each of the four bases in RNA is susceptible to chemical modification in a different way. In this protocol, end-labeled RNAs are subjected to base-specific chemical modification reactions that make the RNA strand adjacent to the modified base susceptible to cleavage. The chemical modification reaction is base-specific but limited so that not every base in every strand is modified. After cleavage, the resulting set of radioactive fragments is resolved via polyacrylamide gel electrophoresis.

INTRODUCTIONThis article describes the analysis and quantification of Arabidopsis RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RNA is first reverse-transcribed into cDNA, which is then amplified by competitive PCR using a mimic. Multiple PCRs are performed using a constant amount of unknown cDNA and varying amounts of a mimic; that is, a template of known concentration that is close in size and GC content to the quantified target and that contains the same annealing sites for primers. Mimics are usually designed to produce PCR products ~10%-20% smaller or larger than the endogenous target. A mimic can be used as a DNA standard (as in this protocol), to be included after the reverse transcription reaction, or, even better, as an RNA standard. In the latter case, an RNA molecule must be generated from a vector designed for in vitro transcription, such as pGEM or pBluescript series vectors. The procedure described here is based on commercial kit protocols.

INTRODUCTIONThis article describes the analysis of Arabidopsis RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RNA is first reverse-transcribed into cDNA, which is then amplified by PCR. This method is quick and easy. Although RT-PCR can be performed quantitatively, it is often technically challenging to obtain highly accurate results. We recommend the use of an RT-PCR kit; the procedure given here is adapted from a commercial kit protocol. In the PCR step, it is advisable to use oligonucleotide primers that span at least one intron. In this way, amplification of any contaminating genomic DNA is readily detected, because the product derived from genomic DNA will be larger than the cDNA product. Expression levels are determined by comparing against a gene that is expressed fairly uniformly, such as the ubiquitin gene UBQ10.

RNA sequencing (RNA-Seq) uses the capabilities of high-throughput sequencing methods to provide insight into the transcriptome of a cell. Compared to previous Sanger sequencing- and microarray-based methods, RNA-Seq provides far higher coverage and greater resolution of the dynamic nature of the transcriptome. Beyond quantifying gene expression, the data generated by RNA-Seq facilitate the discovery of novel transcripts, identification of alternatively spliced genes, and detection of allele-specific expression. Recent advances in the RNA-Seq workflow, from sample preparation to library construction to data analysis, have enabled researchers to further elucidate the functional complexity of the transcription. In addition to polyadenylated messenger RNA (mRNA) transcripts, RNA-Seq can be applied to investigate different populations of RNA, including total RNA, pre-mRNA, and noncoding RNA, such as microRNA and long ncRNA. This article provides an introduction to RNA-Seq methods, including applications, experimental design, and technical challenges.

INTRODUCTIONThe crucial roles of RNA-binding proteins in all aspects of RNA metabolism, particularly in the regulation of mRNAs and subsequent control of gene expression patterns, have become increasingly evident. RNA immunoprecipitation (RIP) is a powerful technique used to detect the association of individual proteins with specific RNA molecules in vivo. Live cells are treated with formaldehyde to generate protein-RNA cross-links between proximal molecules. Following immunoprecipitation of a protein of interest and cross-link reversal, associated RNAs can be recovered, characterized, and quantitated by reverse transcriptase polymerase chain reaction (RT-PCR). Protein association with specific RNA regions can be performed under a variety of conditions (e.g., different environmental and cell-cycle states) and/or in mutant strains. Furthermore, because formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, RIP provides snapshots of protein-RNA interactions at specific time points and hence is useful for kinetic analyses of events occurring on RNA in vivo. The basics of RIP are very similar to chromatin immunoprecipitation (ChIP), but with some caveats that are important to appreciate to take full advantage of the possibilities afforded by RIP.