TY - JOUR
T1 - Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation
AU - Kofoed, Rikke H
AU - Betzer, Cristine
AU - Lykke-Andersen, Søren
AU - Molska, Ewa
AU - Jensen, Poul H
PY - 2018/5/3
Y1 - 2018/5/3
N2 - When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.
AB - When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.
KW - 5-Bromouridine labelling
KW - Genetics
KW - Immunoprecipitation
KW - Issue 135
KW - Phenol-chloroform extraction
KW - Quantitative polymerase chain reaction
KW - RNA synthesis
KW - Reverse transcription
U2 - 10.3791/57056
DO - 10.3791/57056
M3 - Journal article
C2 - 29782024
SN - 1940-087X
VL - 2018
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 135
M1 - e57056
ER -