Nuclear run-on

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A nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain time point. Approximately one million cell nuclei are isolated and incubated with labeled nucleotides, and genes in the process of being transcribed are detected by hybridization of extracted RNA to gene specific probes on a blot. [1] Garcia-Martinez et al. (2004) [2] developed a protocol for the yeast S. cerevisiae (Genomic run-on, GRO) that allows for the calculation of transcription rates (TRs) for all yeast genes to estimate mRNA stabilities for all yeast mRNAs. [3]

Alternative microarray methods have recently been developed, mainly PolII RIP-chip: RNA immunoprecipitation of RNA polymerase II with phosphorylated C-terminal domain directed antibodies and hybridization on a microarray slide or chip (the word chip in the name stems from "ChIP-chip" where a special Affymetrix GeneChip was required). A comparison of methods based on run-on and ChIP-chip has been made in yeast (Pelechano et al., 2009). A general correspondence of both methods has been detected but GRO is more sensitive and quantitative. It has to be considered that run-on only detects elongating RNA polymerases whereas ChIP-chip detects all present RNA polymerases, including backtracked ones.

Overview of the Global run on sequencing assay for delineating genes, genome-wide, that are engaged in transcription. GRO-Seq.png
Overview of the Global run on sequencing assay for delineating genes, genome-wide, that are engaged in transcription.

Attachment of new RNA polymerase to genes is prevented by inclusion of sarkosyl. Therefore only genes that already have an RNA polymerase will produce labeled transcripts. RNA transcripts that were synthesized before the addition of the label will not be detected as they will lack the label. These run on transcripts can also be detected by purifying labeled transcripts by using antibodies that detect the label and hybridizing these isolated transcripts with gene expression arrays or by next generation sequencing (GRO-Seq). [4]

Run on assays have been largely supplanted with Global Run on assays that use next generation DNA sequencing as a readout platform. These assays are known as GRO-Seq and provide an incredibly detailed view of genes engaged in transcription with quantitative levels of expression. Array based methods for analyzing Global run on (GRO) assays are being replaced with Next Generation Sequencing which eliminates the design of probes against gene sequences. Sequencing will catalog all transcripts produced even if they are not reported in databases. GRO-seq involves the labeling of newly synthesized transcripts with bromouridine (BrU). Cells or nuclei are incubated with BrUTP in the presence of sarkosyl, which prevents the attachment of RNA polymerase to the DNA. Therefore only RNA polymerase that are already on the DNA before the addition of sarkosyl will produce new transcripts that will be labeled with BrU. The labeled transcripts are captured with anti-BrU antibody labeled beads, converted to cDNAs and then sequenced by Next Generation DNA sequencing. The sequencing reads are then aligned to the genome and number of reads per transcript provide an accurate estimate of the number of transcripts synthesized. [5]

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Complementary DNA Single-stranded DNA synthesized from RNA

In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists want to express a specific protein in a cell that does not normally express that protein, they will transfer the cDNA that codes for the protein to the recipient cell. In molecular biology, cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays and RNA-seq.

Transcription (biology) Process of copying a segment of DNA into RNA

Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). Averaged over multiple cell types in a given tissue, the quantity of mRNA is more than 10 times the quantity of ncRNA. The general preponderance of mRNA in cells is valid even though less than 2% of the human genome can be transcribed into mRNA, while at least 80% of mammalian genomic DNA can be actively transcribed, with the majority of this 80% considered to be ncRNA.

The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The term transcriptome is a portmanteau of the words transcript and genome; it is associated with the process of transcript production during the biological process of transcription.

This is a list of topics in molecular biology. See also index of biochemistry articles.

<i>In situ</i> hybridization

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

RNA spike-in

An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.

ChIP-on-chip Molecular biology method

ChIP-on-chip is a technology that combines chromatin immunoprecipitation ('ChIP') with DNA microarray ("chip"). Like regular ChIP, ChIP-on-chip is used to investigate interactions between proteins and DNA in vivo. Specifically, it allows the identification of the cistrome, the sum of binding sites, for DNA-binding proteins on a genome-wide basis. Whole-genome analysis can be performed to determine the locations of binding sites for almost any protein of interest. As the name of the technique suggests, such proteins are generally those operating in the context of chromatin. The most prominent representatives of this class are transcription factors, replication-related proteins, like origin recognition complex protein (ORC), histones, their variants, and histone modifications.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

Tiling array

Tiling arrays are a subtype of microarray chips. Like traditional microarrays, they function by hybridizing labeled DNA or RNA target molecules to probes fixed onto a solid surface.

RNA immunoprecipitation chip

RIP-chip is a molecular biology technique which combines RNA immunoprecipitation with a microarray. The purpose of this technique is to identify which RNA sequences interact with a particular RNA binding protein of interest in vivo. It can also be used to determine relative levels of gene expression, to identify subsets of RNAs which may be co-regulated, or to identify RNAs that may have related functions. This technique provides insight into the post-transcriptional gene regulation which occurs between RNA and RNA binding proteins.

Epigenomics is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome. The field is analogous to genomics and proteomics, which are the study of the genome and proteome of a cell. Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene expression without altering the DNA sequence. Epigenomic maintenance is a continuous process and plays an important role in stability of eukaryotic genomes by taking part in crucial biological mechanisms like DNA repair. Plant flavones are said to be inhibiting epigenomic marks that cause cancers. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Epigenetic modifications play an important role in gene expression and regulation, and are involved in numerous cellular processes such as in differentiation/development and tumorigenesis. The study of epigenetics on a global level has been made possible only recently through the adaptation of genomic high-throughput assays.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes. ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers. ChIP is crucial for the advancements in the field of epigenomics and learning more about epigenetic phenomena.

ChIP-exo

ChIP-exo is a chromatin immunoprecipitation based method for mapping the locations at which a protein of interest binds to the genome. It is a modification of the ChIP-seq protocol, improving the resolution of binding sites from hundreds of base pairs to almost one base pair. It employs the use of exonucleases to degrade strands of the protein-bound DNA in the 5'-3' direction to within a small number of nucleotides of the protein binding site. The nucleotides of the exonuclease-treated ends are determined using some combination of DNA sequencing, microarrays, and PCR. These sequences are then mapped to the genome to identify the locations on the genome at which the protein binds.

Single cell sequencing examines the sequence information from individual cells with optimized next-generation sequencing (NGS) technologies, providing a higher resolution of cellular differences and a better understanding of the function of an individual cell in the context of its microenvironment. For example, in cancer, sequencing the DNA of individual cells can give information about mutations carried by small populations of cells. In development, sequencing the RNAs expressed by individual cells can give insight into the existence and behavior of different cell types. In microbial systems, a population of the same species can appear to be genetically clonal, but single-cell sequencing of RNA or epigenetic modifications can reveal cell-to-cell variability that may help populations rapidly adapt to survive in changing environments.

DRIP-seq (DRIP-sequencing) is a technology for genome-wide profiling of a type of DNA-RNA hybrid called an "R-loop". DRIP-seq utilizes a sequence-independent but structure-specific antibody for DNA-RNA immunoprecipitation (DRIP) to capture R-loops for massively parallel DNA sequencing.

Epitranscriptomic sequencing

In epitranscriptomic sequencing, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the RNA sequencer, or (2) improving or modifying bioinformatics analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for mRNA molecules, except for modified bisulfite sequencing for profiling 5-methylcytidine which was optimized for tRNAs and rRNAs.

Time-resolved RNA sequencing methods are applications of RNA-seq that allow for observations of RNA abundances over time in a biological sample or samples. Second-Generation DNA sequencing has enabled cost effective, high throughput and unbiased analysis of the transcriptome. Normally, RNA-seq is only capable of capturing a snapshot of the transcriptome at the time of sample collection. This necessitates multiple samplings at multiple time points, which increases both monetary and time costs for experiments. Methodological and technological innovations have allowed for the analysis of the RNA transcriptome over time without requiring multiple samplings at various time points.

CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.

CITE-Seq is a method for performing RNA sequencing along with gaining quantitative and qualitative information on surface proteins with available antibodies on a single cell level. So far, the method has been demonstrated to work with only a few proteins per cell. As such, it provides an additional layer of information for the same cell by combining both proteomics and transcriptomics data. For phenotyping, this method has been shown to be as accurate as flow cytometry by the groups that developed it. It is currently one of the main methods, along with REAP-Seq, to evaluate both gene expression and protein levels simultaneously in different species.

CUT&Tag-sequencing, also known as cleavage under targets and tagmentation, is a method used to analyze protein interactions with DNA. CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN and CUT&Tag sequencing do not. CUT&Tag sequencing is an improvement over CUT&RUN because it does not require cells to be lysed or chromatin to be fractionated. CUT&RUN is not suitable for single-cell platforms so CUT&Tag is advantageous for these.

References

  1. ↑ Gariglio, P; Bellard, M; Chambon, P (Jun 1981). "Clustering of RNA polymerase B molecules in the 5' moiety of the adult beta-globin gene of hen erythrocytes". Nucleic Acids Res. 9 (11): 2589–98. doi:10.1093/nar/9.11.2589. PMC   326874 . PMID   6269056.
  2. ↑ García-Martínez, J; Aranda, A; Pérez-Ortín, JE (2004). "Genomic run-on evaluates transcription rates for all yeast genes and identifies gene regulatory mechanisms". Mol. Cell. 15 (2): 303–13. doi:10.1016/j.molcel.2004.06.004. hdl: 10550/32058 . PMID   15260981.
  3. ↑ Pelechano, V; Jimeno-González, S; Rodríguez-Gil, A; García-Martínez, J; Pérez-Ortín, JE; Chávez, S (Aug 2009). "Regulon-specific control of transcription elongation across the yeast genome". PLOS Genet. 5 (8): e1000614. doi:10.1371/journal.pgen.1000614. PMC   2721418 . PMID   19696888.
  4. ↑ Core, L; Waterfall, J; Lis, JT (2008). "Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters". Science. 322 (5909): 1845–8. Bibcode:2008Sci...322.1845C. doi:10.1126/science.1162228. PMC   2833333 . PMID   19056941.
  5. ↑ Min, IM; Waterfall, JJ; Core, LJ; Munroe, RJ; Schimenti, J; Lis, JT (Apr 2011). "Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells". Genes Dev. 25 (7): 742–54. doi:10.1101/gad.2005511. PMC   3070936 . PMID   21460038.
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