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
<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Regulation+and+its+Misregulation+in+Disease.">Transcriptional Regulation and its Misregulation in Disease.</a> Cell. 152, 1237-1251 (2014).</li><li>Farrer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+kindreds+with+parkinsonism+and+&#945;-synuclein+genomic+multiplications.">Comparison of kindreds with parkinsonism and &#945;-synuclein genomic multiplications.</a> Ann. Neurol. 55, 174-179 (2004).</li><li>Khodr, C. E., Becerra, A., Han, Y., Bohn, M. 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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 ...

The overall goal of this experiment is to measure changes in RNA synthesis by bromouridine labeling and immunoprecipitation. This method can help answer key questions in the field of RNA metabolism by measuring whether a change in RNA levels is caused by changes in RNA synthesis. The main advantage of this technique is that it can be used on any preferred type of cell culture and the materials used are easily accessible.To begin the protocol, seed 3.5 times 10 to the sixth HEK293T cells in a 10-centimeter Petri dish in 10 milliliters of growth media to obtain a total of greater than 40 micrograms of RNA upon extraction 48 hours later. Maintain the cells at 37 degrees Celsius and 5%CO2.48 hours after seeding, aspirate eight milliliters of growth media, and leave two milliliters behind to prevent the cells from drying out. Add two millimoles of bromouridine and treatment to the growth media, and remove the residual two milliliters from the cells before reapplying the eight milliliters of bromouridine containing growth media.Next, incubate the cells for one hour. After incubation, wash the cells in five milliliters of Hank's buffer, and trypsinize the cells with two milliliters of 0.05%trypsin for five minutes. Add eight milliliters of growth media to stop the reaction, transfer the cells to a 15-milliliter tube, and spin the cells for two minutes at 1, 000 times g.Then, remove the supernatant and extract total RNA from the cell pellet using an RNA isolation kit, and elute the RNA in RNase-free water. This technique is very sensitive, and it's therefore important to work with RNA of a high quality. The RNA extraction is therefore a critical step, but it's also easily performed with today's RNA extraction kits.Prepare beads in one batch for the number of samples to be investigated plus one extra to account for loss during pipetting and whirl mixing. Resuspend anti-Mouse IgG magnetic beads thoroughly by whirl mixing, and take out 20 microliters of beads per sample in an RNase-free 1.5-milliliter tube. Place the 1.5-milliliter tube in a magnetic stand for 20 seconds for the beads to collect on the side.Remove the supernatant, take out the tube from the magnetic stand, and resuspend the beads in 40 microliters of one times bromouridine-IP buffer by pipetting. Spin the tube for two seconds in a tabletop centrifuge, place it back in the magnetic stand, and remove the supernatant. Next, block unspecific binding to the beads using heparin.Resuspend the beads in one milliliter of one times bromouridine-IP plus one milligram per milliliter of heparin buffer, and rotate the tube for 30 minutes. Then, wash the beads one time. Resuspend the beads in one milliliter of one times bromouridine-IP buffer, and add 1.25 micrograms per sample of anti-alpha-bromodeoxyuridine antibody.Incubate the beads for one hour while rotating. Following incubation, wash the beads three times. Resuspend the washed beads in 50 microliters per sample of one times bromouridine-IP buffer supplemented with one millimolar bromouridine, and leave the beads rotating for 30 minutes.After rotation, wash the beads three times, and resuspend the beads in 50 microliters per sample of one times bromouridine-IP buffer. Measure RNA concentrations in the RNA extracts using ultraviolet-visible spectrophotometry, and dilute 40 micrograms of RNA from each sample in RNase-free water to a total volume of 200 microliters. After denaturing the RNA for two minutes at 80 degrees Celsius, spin the samples briefly in a tabletop centrifuge.After spinning, add 200 microliters of two times bromouridine-IP plus BSA, RNase inhibitor. Then, add 50 microliters of the resuspended and prepared beads to each sample, and rotate the samples for one hour. After incubation, wash the beads four times.Resuspend the beads in 200 microliters of elution buffer to elute the RNA, and quickly add 200 microliters of pH 6.6 phenol to remove any non-RNA molecules from the sample. Whirl the sample to mix, and spin it for three minutes at 25, 000 times g in a tabletop centrifuge. Next, aspirate as much of the upper phase as possible, approximately 190 microliters, and move it to a tube with 200 microliters of pH 6.6 phenol-chloroform.Make sure to aspirate the same amount across samples. Whirl the sample, and spin it. After the spin, perform chloroform extraction by aspirating the aqueous upper phase, approximately 180 microliters, and transfer it to a tube containing 200 microliters of chloroform.Whirl and then spin the sample. Aspirate the aqueous upper phase, approximately 170 microliters, and transfer it to a tube containing 17 microliters of three molar pH six sodium acetate to neutralize and precipitate the RNA from the aqueous solution. Next, add two microliters of glycogen, which will precipitate with the RNA and make the pellet more visible.Add 500 microliters of 96%ethanol to increase binding between the salt ions and the RNA, and mix by inverting the tube a couple of times. Leave the tube overnight at minus 20 degrees Celsius or for 15 minutes on dry ice. Spin the chilled sample, discard supernatant without disturbing the pellet, and wash the pellet in 185 microliters of 75%ethanol to remove any residual salt.Then, spin the sample at 25, 000 times g at four degrees Celsius for five minutes. Discard the supernatant completely without disturbing the pellet, and leave the pellet to dry for five to 10 minutes with the lid open. Resuspend the pellet in 10 microliters of RNase-free water applied on the side of the tube to avoid disturbing the pellet.AsPC-1 cells were treated with bromouridine for zero, one, two, and 4.5 hours, followed by total RNA extraction and bromouridine-IP. GAS5 and GAPDH were measured in bromouridine-IP RNA, which demonstrated that one hour of labeling was sufficient to reach nine-and 44-fold change compared to the background. Two millimoles of bromouridine treatment did not induce any morphologic changes in HEK293T cells for either one hour or the longer four-hour treatment.PLK-2 inhibition caused an increase in alpha-synuclein mRNA in both the input and bromouridine-IP, suggesting that the increase in the steady state RNA is caused by an increase in synthesis of RNA. After watching this video, you should have a good understanding of how to label newly synthesized RNA with bromouridine and perform a subsequent immunoprecipitation of bromouridine and the labeled RNA.

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