Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Summary

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

Abstract

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.

Introduction

5-Bromouridine (BrU) immunoprecipitation (IP) allows the study of RNA production in cells with no or very limited effects on cell physiology during the brief labelling period^1,2. The method is based upon incorporation of the synthetic uridine derivative BrU into newly synthesized RNA followed by IP of labelled RNA using anti-BrU antibodies (Figure 1).

It has been known for decades that protein synthesis can be transcriptionally regulated, and the existence of transcription factors was hypothesized more than 50 years ago³. Today, ....

Protocol

NOTE: Perform all steps at room temperature unless otherwise stated and keep buffers on ice or in the fridge (except from the Elution buffer containing SDS). The protocol is divided into 5 sections: Preparation of RNA samples; preparation of beads for IP; binding of BrU-labelled RNA to beads; elution and purification of bound BrU-labelled RNA by 3 steps phenol-phenol/chloroform-chloroform extraction; analysis of RNA (in this example using RT-qPCR)

1. Preparation of RNA Samples

1. Count HEK293T cells using an automated cell counter and seed 3,500,000 cells in a 10 cm petri dish in 10 mL growth media (Dulbecco's Modified Eagle....

Results

Our initial tests of BrU-labelling time were performed in AsPC-1 cells. The cells were treated with BrU for 0, 1, 2, and 4.5 h, followed by total RNA extraction and BrU-IP. GAS5 and GAPDH were measured in BrU-IP RNA, which demonstrated that 1 h labelling was sufficient to reach an approximately 9- and 44-fold change compared to background (0 h) for GAPDH and GAS5, respectively.

BrU-IP and the analysis described above were used t.......

Discussion

This protocol describes the use of BrU-IP for determination of RNA production by labelling of newly produced RNA for 1 h with BrU, followed by immediate RNA extraction and immunoprecipitation of BrU-labelled RNA. This method has previously been described in reference¹⁶, and this article includes some additional steps to increase IP specificity and RNA purity, by pre-treating beads with low concentrations of BrU as well as a phenol-chloroform purification step to increase RNA purity following IP. A.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ribolock Rnase inhibitor	ThermoFisher	EO0381
Dynabeads M-280 Sheep anti-mouse IgG	Life Technologies	11202D
α-Bromodeoxyuridine mouse antibody	BD Bioscience	555627
Nanodrop 1000	Thermo Fisher		Ultraviolet-visible spectrophotometry
Water-Saturated Phenol pH 6.6	ThermoFisher	AM9712
Phenol:Chloroform:IAA 25:24:1 pH 6.6	ThermoFisher	AM9732
High Pure RNA isolation Kit	Roche	11828665001
High Capacity cDNA Reverse Transcription Kit	ThermoFisher	4368814
Taqman Fast Advanced Master Mix	ThermoFisher	4444557	qPCR Master Mix
Taqman RNA probes	ThermoFisher	SNCA: Hs01103383_m1, 18sRNA: Hs03928985_g1, ACTB: Hs01060665_g1, HMBS: Hs00609296, NADH: Hs01072843_m1	Flurogenic probes
7500 Fast Real-Time PCR system	Applied Biosystems
HEK293T cell line	ATCC
Dulbecco's Modified Eagle's Medium	Lonza	BE12-604F
Fetal calf serum	Biochrom	S0115
Penicillin/Streptomycin	Biochrom	A2213
Hank's buffer			5,3 mM KCl, 0.44 mM KH2PO4, 0.2 mM Na2HPO4 and 136.8 mM NaCl pH 6.77, autoclaved.
DEPC-H2O			0.1% diethylpyrocarbonate treated H2O
2xBrU-IP Buffer			40 mM Tris-HCl pH 7.5 and 500 mM NaCl in DEPC H2O
2xBrU-IP+BSA/RNAse inhibitor			2xBrU-IP buffer supplemented with 1 μg/μL BSA and 80 U/mL Ribolock
BrU-IP+ 1 mg/mL heparin			2xBrU-IP buffer diluted 1:1 DEPC-H2O and supplemented with 1mg/mL heparin
1xBrU-IP			2xBrU-IP buffer diluted 1:1 in DEPC-H2O and supplemented with 0.5 μg/μL BSA and 20 U/mL Ribolock
Elution buffer			0.1% SDS in RNAse-free H2O