Name: investigation of rna synthesis using 5-bromouridine labelling and immunoprecipitation
Uploaded: 2018-05-03T13:00:00
Description: Watch this Scientific Journal Video about Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation at JoVE.com

The Lundbeck Foundation, Aarhus University, Dandrite and Lundbeck A/S supported this work.

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
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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...

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 period1,2. 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, it is known that several diseases are caused by dysregulation of transcription and RNA stability (Supplementary Figure S1 in reference4), and the ability to measure changes in RNA production is of great importance in understanding disease development.
On the other hand, tools to regulate RNA production provide new possibilities for treating diseases caused by too little or too much protein expression, or protein accumulation such as in Parkinson&#39;s disease (PD). The predominant protein accumulating as insoluble aggregates in PD is &#945;-synuclein, and the level of &#945;-synuclein is directly linked to the disease, as gene multiplication of the &#945;-synuclein gene causes familial PD5. Furthermore, &#945;-synuclein aggregates in a concentration dependent manner. Downregulation of &#945;-synuclein mRNA levels is therefore an interesting therapeutic strategy, which has been successfully achieved using RNA interference to decrease neuronal cell loss in PD rodent models6,7.
It is important to be able to measure the changes in RNA production caused by either disease or therapeutic interventions. State of the art methods for measuring steady state levels of RNA, such as reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), are not capable of distinguishing between changes caused by transcriptional regulation or by altered RNA stability. A widely used method for investigation of RNA decay rates is blocking of the transcriptional machinery using compounds such as &#945;-amanitin or actinomycin D followed by measurements of the decaying RNAs. However, there are some problems associated with an overall blockage of transcription in cells, such as induction of apoptosis8,9. Beside the cytotoxic effects, transcriptional inhibitors also present several technical issues, such as slow cellular uptake of &#945;-amanitin and lack of specificity of actinomycin D (reviewed in reference10).
To avoid the overall blockage of transcription, nucleotide analogues have been used, such as 5-ethynyl uridine (5-EU), 4-thiouridine (4-TU), and BrU. These are readily taken up by mammalian cells and incorporated into newly synthesized RNA, enabling pulse-labelling of RNA within a specific time-frame. BrU is less toxic than 5-EU and 4-TU, making it the preferred analogue of choice11,12.
BrU-IP can be used to investigate both the rate of RNA synthesis and stability, and thus distinguish between the underlying causes of changes in total RNA. This article will focus on the measurement of RNA synthesis and refers to reference2 for details on investigation of RNA stability. To investigate RNA synthesis, cells are briefly labelled with BrU e.g. for 1 h followed by BrU-IP1 (Figure 1C). This enables measurements of RNA synthesized within the short labelling time and observed changes will give a better estimate of regulations in RNA synthesis than by measuring changes in total RNA by RT-qPCR. It should be mentioned, however, that even though the labelling time is, for example, only 1 h, degradation can still have an influence on the RNA levels observed.
RNA from BrU-IP experiments are suitable for downstream analysis by both RT-qPCR1 or next generation sequencing2,13. Other standard RNA detection methods, such as northern blotting or ribonuclease protection assay, may also be applicable for certain RNAs.

<table>
	<tbody>
		<tr>
			<td>Ribolock Rnase inhibitor</td>
			<td>ThermoFisher</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 Sheep anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>11202D</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Bromodeoxyuridine mouse antibody</td>
			<td>BD Bioscience</td>
			<td>555627</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 1000</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>Ultraviolet-visible spectrophotometry</td>
		</tr>
		<tr>
			<td>Water-Saturated Phenol pH 6.6</td>
			<td>ThermoFisher</td>
			<td>AM9712</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA 25:24:1 pH 6.6</td>
			<td>ThermoFisher</td>
			<td>AM9732</td>
			<td></td>
		</tr>
		<tr>
			<td>High Pure RNA isolation Kit</td>
			<td>Roche</td>
			<td>11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Advanced Master Mix</td>
			<td>ThermoFisher</td>
			<td>4444557</td>
			<td>qPCR Master Mix</td>
		</tr>
		<tr>
			<td>Taqman RNA probes</td>
			<td>ThermoFisher</td>
			<td>SNCA: Hs01103383_m1, 18sRNA: Hs03928985_g1, ACTB: Hs01060665_g1, HMBS: Hs00609296, NADH: Hs01072843_m1</td>
			<td>Flurogenic probes</td>
		</tr>
		<tr>
			<td>7500 Fast Real-Time PCR system</td>
			<td colspan="2">Applied Biosystems</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cell line</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Biochrom</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom</td>
			<td>A2213</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s buffer</td>
			<td></td>
			<td></td>
			<td>5,3 mM KCl, 0.44 mM KH2PO4, 0.2 mM Na2HPO4 and 136.8 mM NaCl pH 6.77, autoclaved.</td>
		</tr>
		<tr>
			<td>DEPC-H2O</td>
			<td></td>
			<td></td>
			<td>0.1% diethylpyrocarbonate treated H2O</td>
		</tr>
		<tr>
			<td>2xBrU-IP Buffer</td>
			<td></td>
			<td></td>
			<td>40 mM Tris-HCl pH 7.5 and 500 mM NaCl in DEPC H2O</td>
		</tr>
		<tr>
			<td>2xBrU-IP+BSA/RNAse inhibitor</td>
			<td></td>
			<td></td>
			<td>2xBrU-IP buffer supplemented with 1 &#956;g/&#956;L BSA and 80 U/mL Ribolock</td>
		</tr>
		<tr>
			<td>BrU-IP+ 1 mg/mL heparin</td>
			<td></td>
			<td></td>
			<td>2xBrU-IP buffer diluted 1:1 DEPC-H2O and supplemented with 1mg/mL heparin</td>
		</tr>
		<tr>
			<td>1xBrU-IP</td>
			<td></td>
			<td></td>
			<td>2xBrU-IP buffer diluted 1:1 in DEPC-H2O and supplemented with 0.5 &#956;g/&#956;L BSA and 20 U/mL Ribolock</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td></td>
			<td></td>
			<td>0.1% SDS in RNAse-free H2O</td>
		</tr>
	</tbody>
</table>

investigation of rna synthesis using 5-bromouridine labelling and immunoprecipitation

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 &#945;-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.

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...

Watch this Scientific Journal Video about Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation at JoVE.com

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

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.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

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