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== Measuring and detecting == [[File:Transcription label en.jpg|thumb| [[Electron micrograph]] of transcription of ribosomal RNA. The forming [[ribosomal RNA]] strands are visible as branches from the main DNA strand.{{citation needed|date=January 2011}} ]] Transcription can be measured and detected in a variety of ways:{{citation needed|date=January 2011}} * [[G-Less Cassette]] transcription assay: measures promoter strength * [[Run-off transcription]] assay: identifies transcription start sites (TSS) * [[Nuclear run-on]] assay: measures the relative abundance of newly formed transcripts * [[KAS-seq]]: measures single-stranded DNA generated by RNA polymerases; can work with 1,000 cells.<ref>{{cite journal | vauthors = Wu, T| title = Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ | journal = Nature Methods | volume = 17 | issue = 5 | pages = 515β523 | date = April 2020 | doi = 10.1038/s41592-020-0797-9 | pmid = 32251394 | s2cid = 214810294 | pmc = 7205578 }}</ref> * [[RNase protection assay]] and [[ChIP-Chip]] of [[RNAP]]: detect active transcription sites * [[RT-PCR]]: measures the absolute abundance of total or nuclear RNA levels, which may however differ from transcription rates * [[DNA microarrays]]: measures the relative abundance of the global total or nuclear RNA levels; however, these may differ from transcription rates * [[In situ hybridization]]: detects the presence of a transcript * [[MS2 tagging]]: by incorporating RNA [[Stem-loop|stem loops]], such as MS2, into a gene, these become incorporated into newly synthesized RNA. The stem loops can then be detected using a fusion of GFP and the MS2 coat protein, which has a high affinity, sequence-specific interaction with the MS2 stem loops. The recruitment of GFP to the site of transcription is visualized as a single fluorescent spot. This new approach has revealed that transcription occurs in discontinuous bursts, or pulses (see [[Transcriptional bursting]]). With the notable exception of in situ techniques, most other methods provide cell population averages, and are not capable of detecting this fundamental property of genes.<ref>{{cite journal | vauthors = Raj A, van Oudenaarden A | title = Nature, nurture, or chance: stochastic gene expression and its consequences | journal = Cell | volume = 135 | issue = 2 | pages = 216β26 | date = October 2008 | pmid = 18957198 | pmc = 3118044 | doi = 10.1016/j.cell.2008.09.050 }}</ref> * [[Northern blot]]: the traditional method, and until the advent of [[RNA-Seq]], the most quantitative * [[RNA-Seq]]: applies next-generation sequencing techniques to sequence whole [[transcriptome]]s, which allows the measurement of relative abundance of RNA, as well as the detection of additional variations such as fusion genes, post-transcriptional edits and novel splice sites * [[Single-cell RNA-sequencing|Single cell RNA-Seq]]: amplifies and reads partial transcriptomes from isolated cells, allowing for detailed analyses of RNA in tissues, embryos, and cancers
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