Nuclear run-on
Encyclopedia
A nuclear run-on assay is conducted to identify the gene
s that are being transcribed
at a certain time. Cell nuclei
are isolated rapidly, and incubated with labelled nucleotide
s and the results are hybridized to a slot blot, which is then exposed to film. It was originally developed by Gariglio et al. (1981) and Brown et al. (1984) (see discussion).
Adding actinomycin-D to the reaction buffer is sufficient to stop transcription, while other toxins, such as α-amanitin, show different effects, making it a good way to assay the effects of these compounds.
This method allows changes in transcription
rates to be measured, which often differ from steady-state mRNA levels used in microarrays. Although the method is time-consuming and requires the use of radioactivity and large numbers of cells, it remains the most reliable method to measure transcription rates directly. Several attempts recently have been made to make this method microarray-compatible, but other methods are much more favorable. To have a publication quality blot about 107 nuclei are needed, but 106 nuclei are sufficient to give results: these quantities of cells are higher than for most protocols.
It is often cited that this method has been surpassed by microarrays, even though this method compares transcription rates and not total transcript expression levels, which differ.
It is incompatible with microarray experiments due to the fact eukaryotic polymerases are more intolerant towards fluorescently labeled or aminoallyl nucleotides, so UTP with phosphorus-32, or phosphorus-33, on the alpha phosphate group is normally used. Cheadle et al. (2005) used spotted nylon blots to obtain a high-throughput analysis of T-cell activation. Garcia-Martinez et al (2004)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. Those authors also used the calculated TRs and the steady-state mRNA amounts of all genes to determine, at global scale, the mRNA stabilities for all yeast mRNAs.
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.
The main issue on the validity of run on assay results is the presence of artifacts due to the isolation step: some transcripts may stop prematurely others might start out of place.
Another point to add is about the nuclei: There are various methods that require nuclear isolation. The problem is that the nuclear membrane is contiguous with the ER which is rich in protein and cytoplasmic mRNA; some methods require pure nuclear lysate while others, like a nuclear run-on, require "dirty" nuclei with an intact nuclear membrane even if the ER is attached. Some types of cell are notoriously difficult to isolate this way such as lymphocytes, which require a sucrose gradient purification. Yeast cells do not require nuclei isolation. Permeabilized cells (with sarkosil) can be used. This is a clear technical advantage.
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s that are being transcribed
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
at a certain time. Cell nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
are isolated rapidly, and incubated with labelled nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s and the results are hybridized to a slot blot, which is then exposed to film. It was originally developed by Gariglio et al. (1981) and Brown et al. (1984) (see discussion).
Adding actinomycin-D to the reaction buffer is sufficient to stop transcription, while other toxins, such as α-amanitin, show different effects, making it a good way to assay the effects of these compounds.
This method allows changes in transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
rates to be measured, which often differ from steady-state mRNA levels used in microarrays. Although the method is time-consuming and requires the use of radioactivity and large numbers of cells, it remains the most reliable method to measure transcription rates directly. Several attempts recently have been made to make this method microarray-compatible, but other methods are much more favorable. To have a publication quality blot about 107 nuclei are needed, but 106 nuclei are sufficient to give results: these quantities of cells are higher than for most protocols.
It is often cited that this method has been surpassed by microarrays, even though this method compares transcription rates and not total transcript expression levels, which differ.
It is incompatible with microarray experiments due to the fact eukaryotic polymerases are more intolerant towards fluorescently labeled or aminoallyl nucleotides, so UTP with phosphorus-32, or phosphorus-33, on the alpha phosphate group is normally used. Cheadle et al. (2005) used spotted nylon blots to obtain a high-throughput analysis of T-cell activation. Garcia-Martinez et al (2004)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. Those authors also used the calculated TRs and the steady-state mRNA amounts of all genes to determine, at global scale, the mRNA stabilities for all yeast mRNAs.
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.
The main issue on the validity of run on assay results is the presence of artifacts due to the isolation step: some transcripts may stop prematurely others might start out of place.
Another point to add is about the nuclei: There are various methods that require nuclear isolation. The problem is that the nuclear membrane is contiguous with the ER which is rich in protein and cytoplasmic mRNA; some methods require pure nuclear lysate while others, like a nuclear run-on, require "dirty" nuclei with an intact nuclear membrane even if the ER is attached. Some types of cell are notoriously difficult to isolate this way such as lymphocytes, which require a sucrose gradient purification. Yeast cells do not require nuclei isolation. Permeabilized cells (with sarkosil) can be used. This is a clear technical advantage.