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>
	<li>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&#39;s Modified Eagle&#39;s Medium supplemented with 10% fetal calf serum and 50 &#181;g/mL penicillin/streptomycin) to obtain a total of &#62;40 &#181;g RNA upon extraction 48 h later. Maintain cells at 37 &#176;C and 5% CO2.</li>
	<li>48 h after seeding, aspirate 8 mL growth media and leave 2 mL behind to avoid drying out the cells. Add 2 mM BrU and any treatment to the 8 mL aspirated growth media, and remove the residual 2 mL from the cells before reapplying the 8 mL BrU containing growth media. Avoid the use of fresh media upon application of the BrU to the cells, since this can induce changes in gene expression.</li>
	<li>Incubate the cells for 1 h with BrU and treatment. Wash the cells in 5 mL Hank&#39;s buffer and trypsinize the cells using 2 mL 0.05% trypsin. Add 8 mL growth media to stop the reaction, transfer the cells to a 15 mL tube, and spin down the cells for 2 min at 1,000 x g.</li>
	<li>Remove the supernatant and extract total RNA from the cell pellet using an RNA isolation kit (see Table of Materials) or any other preferred method, and elute in RNase-free H2O. Store the RNA at -80 &#176;C until ready to proceed.</li>
</ol>
2. Preparation of Beads for IP
<ol>
	<li>Prepare beads in batches 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 &#181;L beads per sample in an RNase-free 1.5 mL tube. Place the 1.5 mL tube in a magnetic stand and leave approximately 20 s for the beads to collect on the side.</li>
	<li>Remove the supernatant, take out the tube from the magnetic stand, and resuspend in 400 &#181;L 1x BrU-IP buffer by pipetting. Spin the tube very briefly for 2 s in a table-top centrifuge, place it back in the magnetic stand, and remove the supernatant. Repeat the washing step once.</li>
	<li>Block unspecific binding to the beads using heparin. Resuspend the beads in 1 mL 1x BrU-IP+ 1 mg/mL heparin buffer, and leave rotating for 30 min. Wash the beads once, as in step 2.2. 
	NOTE: Heparin is a negatively charged polymer, which will compete with RNAs for binding. tRNA or other irrelevant RNAs can also be used if the downstream application is not sequencing.</li>
	<li>Resuspend the beads in 1 mL 1x BrU-IP buffer and add 1.25 &#181;g/sample anti-&#945;-Bromodeoxyuridine antibody, which also recognizes BrU. Incubate the beads for 1 h while rotating, or overnight at room temperature.</li>
	<li>Wash the beads three times as in step 2.2. Resuspend the beads in 50 &#181;L/sample 1x BrU-IP buffer supplemented with 1 mM BrU and leave rotating for 30 min to lower the sensitivity of the antibody bound to the beads and hereby the risk of unspecific binding during IP.</li>
	<li>Wash the beads three times as in step 2.2, and resuspend in 50 &#181;L/sample 1xBrU-IP buffer.</li>
</ol>
3. Binding of BrU-Labelled RNA to Beads
<ol>
	<li>Measure RNA concentrations in the RNA extracts from step 1.3 using ultraviolet-visible spectrophotometry, and dilute 40 &#181;g RNA from each sample in RNase-free H2O to a total volume of 200 &#181;L.</li>
	<li>Heat RNA samples for 2 min at 80 &#176;C to denature RNA and spin briefly in a tabletop centrifuge. Add 200 &#181;L 2x BrU-IP+BSA/RNAse inhibitor.</li>
	<li>Add 50 &#181;L resuspended and prepared beads from step 2.6 to each sample and leave rotating 1 h. Wash the beads four times as in step 2.2.</li>
</ol>
4. Elution and Purification of RNA by 3 Steps Phenol-phenol/Chloroform-chloroform Extraction
<ol>
	<li>Elute RNA and perform phenol extraction
	<ol>
		<li>Resuspend the beads in 200 &#181;L Elution buffer to elute RNA, and quickly thereafter add 200 &#181;L phenol, pH 6.6 (CAUTION: Toxic, see note below step 4.1.2) to remove any non-RNA molecules from the sample.</li>
		<li>Whirl mix the sample, and spin 3 min at 25,000 x g in a tabletop centrifuge. 
		NOTE: Phenol is toxic and should be handled in a fume hood wearing skin protection. The slightly acidic pH ensures that only RNA, and not DNA, is extracted. The RNA will remain in the upper aqueous phase due to the hydrophilic nature of charges. The hydrophobic cores of proteins will bind phenol and move to the lower organic phenol-phase.</li>
	</ol>
	</li>
	<li>Perform phenol-chloroform extraction
	<ol>
		<li>Aspirate as much upper-phase as possible (approximately 190 &#181;L of the 200 &#181;L, depending on experience in handling) and move it to a tube with 200 &#181;L phenol/chloroform, pH 6.6 (CAUTION: Toxic, see note). Make sure to aspirate the same amount across samples.</li>
		<li>Whirl mix the sample and spin for 3 min at 25,000 x g. 
		NOTE: Chloroform is toxic and should be handled in a fume hood wearing skin protection. Chloroform is added to remove phenol, which can affect the downstream analysis of RNA by inhibiting reverse transcription14 and polymerase chain reactions15.</li>
	</ol>
	</li>
	<li>Perform chloroform extraction by aspirating the aqueous upper phase as before (now approximately 180 &#181;L), and transfer to a tube containing 200 &#181;L chloroform. Whirl mix the sample and spin 3 min at 25,000 x g.</li>
	<li>Aspirate the aqueous upper phase as before (now approximately 170 &#181;L) and transfer it to a tube containing 17 &#181;L (1/10 volume) 3 M CH3COONa, pH 6.0. To neutralize and precipitate RNA from the aqueous solution, add 2 &#181;L glycogen (20 mg/mL), which precipitates together with RNA and makes the RNA pellet more visible.</li>
	<li>Add 500 &#181;L 96% ethanol to increase binding between the salt ions and RNA and mix by inverting the tube a couple of times. Leave the tube overnight at -20 &#176;C or 15 min on dry ice.</li>
	<li>Spin the sample at 25,000 x g, 4 &#176;C for 30 min. Discard supernatant without disturbing the pellet and wash the pellet in 185 &#181;L 75% ethanol to remove any residual salt. Spin at 25,000 x g, 4 &#176;C for 5 min.</li>
	<li>Discard supernatant completely without disturbing the pellet and leave the pellet to dry 5-10 min with an open lid. Resuspend in 10 &#181;L RNase-free H2O applied on the side of the tube to avoid disturbing the pellet.</li>
</ol>
5. Analysis of RNA
<ol>
	<li>Prepare cDNA using a cDNA reverse transcription kit (see Table of Materials) or any other preferred method using 2 &#181;L RNA sample and 1 &#181;g yeast total RNA as carrier RNA for the cDNA synthesis reaction (not used if cDNA is used for next generation sequencing). Use 1 &#181;g RNA without yeast RNA for cDNA preparation of corresponding input.</li>
	<li>Dilute cDNA 1:25 and mix 9 &#181;L diluted cDNA with 10 &#181;L qPCR Master mix and 1 &#181;L fluorogenic probe, both specified in the Table of Materials. 
	NOTE: The cDNA dilution depends on the cDNA preparation and qPCR method used.</li>
	<li>Run the following qPCR program (e.g., on a 7500 Fast Real-time PCR system or equivalent): 2 min at 50 &#176;C, 20 s at 95 &#176;C, 40 cycles of 3 s at 95 &#176;C and 30 s at 60 &#176;C.</li>
</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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The authors have nothing to disclose.

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. Additionally, we also present data here demonstrating the specificity of the BrU-IP by comparing the RNA captured from BrU and non-BrU treated cells. Phenol-chloroform purification is cheap, but considering the low amounts of RNA labelled with BrU, increased signals in the downstream analysis might be obtained by using RNA clean-up kits instead. Our experience is, however, that the most equal extraction of RNA across samples is obtained using phenol-chloroform purification.
BrU-labelling and -IP are, in contrast to analysis of total RNA levels, capable of investigating the underlying causes of changes in steady state RNA levels. The method has no or limited effects on cell physiology, in contrast to the use of transcriptional inhibitors such as &#945;-amanitin and actinomycin D. It has been suggested that comparison of newly synthesized RNA to steady state levels can be used to calculate RNA decay rates17, and the BrU-IP method described here enables simultaneous determination of both RNA synthesis and decay in one assay.
In Figure 5, we demonstrated that RNA is only IP&#39;ed from BrU treated cells, showing that there is indeed BrU incorporated into the RNA in the cell line used here. However, we suggest conducting an initial test of BrU incorporation into RNA in other cell lines before performing the BrU-IP. This could, for example, be done by using anti-BrU antibodies to quantify BrU in total RNA extracts via dot blot or enzyme-linked immunosorbent assay.
Here we have not presented a test of BrU-IP efficiency across samples, but this can be investigated using BrU-labelled spikes in RNA either produced in vitro or from another organism, such as Drosophila melanogaster. This RNA could then also be used in the downstream quantifications and normalizations.
Correct pipetting techniques are crucial when performing highly sensitive quantitative assays, such as BrU-IP and RT-qPCR, and a particular effort should be made to perform equal pipetting across samples. The phenol-chloroform extractions demand some experience, but upon unsatisfactory pipetting, the aqueous and phenol/chloroform phases can be re-mixed and centrifuged again.
This protocol suggests BrU-labelling for 1 h when measuring RNA synthesis. This labelling time enables a rough estimate of changes in transcription, since the majority of human and mice mRNAs have been estimated to have half-life &#62;6 h18,19. Caution should, however, be exercised when interpreting the data obtained, since many RNAs have half-lives &#60;1 h18,19, such as several transcripts induced by Lipid A, which rapidly increase upon stimulation and decline back to base-line levels within 2 h after induction20. To lower the risk of influences from RNA degradation, the BrU-labelling time can be decreased, however, it is also important to label enough RNA to be able to distinguish RNA obtained by BrU-IP from the background (which was obtained after 1 h labelling in our hands (Figure 2)). Another option is to measure both RNA synthesis and decay of the gene of interest using BrU-labelling and IP, the latter method is described in2. The data from these two approaches supplement each other, and if a change in synthesis between two conditions is measured but no change is seen in decay rates, it can be concluded that the measured changes in RNA were due to regulation of synthesis. However, it is important to remember that both techniques only measure RNA functionality by estimating RNA levels and completely neglect to investigate other mechanisms influencing RNA functions such as chemical modifications14.
Since the cells are incubated with BrU for only 1 h, very slowly produced mRNAs can be difficult to measure using the BrU-IP method. This is one point where the use of transcriptional inhibitors such as &#945;-amanitin and actinomycin D might be preferred over BrU-IP, however, they can only be used to measure changes in degradation and not directly measure RNA production. The low levels of BrU-labelled RNA can also be overcome by increasing the incubation time with BrU, though, long incubation times increase the risk of influences by RNA degradation. In that case, it is no longer possible to know whether changes in BrU-labelled RNA are caused by changes in RNA production or decay.
In this example, the downstream analysis of the captured BrU-labelled RNA is performed using RT-qPCR, however, next generation sequencing is also a widely used downstream method to screen for changes in a large pool of RNAs2,13. Similarly, the method is not restricted to analysis of mRNAs, but may analyze all types of RNAs2.

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>&amp;alpha;-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>Applied Biosystems</td><td></td><td></td></tr><tr><td>HEK293T cell line</td><td>ATCC</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;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&#039;s buffer</td><td></td><td></td><td>5,3 mM KCl, 0.44 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 136.8 mM NaCl pH 6.77, autoclaved.</td></tr><tr><td>DEPC-H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>0.1% diethylpyrocarbonate treated H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;mu;g/&amp;mu;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-H&lt;sub&gt;2&lt;/sub&gt;O 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 &amp;mu;g/&amp;mu;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 to investigate if the changes discovered in steady state levels of &#945;-synuclein mRNA upon Polo-like kinase 2 (PLK-2) inhibition were caused by changes in mRNA synthesis1. A 1 h labelling time was chosen because this provides sufficient labelling compared to background (Figure 2) and also allows the treatment time to have an effect. 2 mM BrU treatment did not induce any morphologic changes in HEK293T cells for either 1 h or the longer 4 h treatment (Figure 3A).
&#945;-Synuclein transfected HEK293T cells were treated for 1 h with DMSO or a specific PLK-2 inhibitor (PLK-2i) in the presence of BrU to label freshly synthesized RNA. Cells were hereafter harvested and total RNA was extracted. BrU-labelled RNA produced within the 1 h treatment was collected using BrU-IP (&#34;BrU-IP&#34; in Figure 3B) from the total pool of RNA (&#34;Input&#34; in Figure 3B). The amount of RNA obtained with BrU-IP from 40 &#181;g starting material varies between different cell lines, and we generally obtain approximately 500 ng from HEK293T cells, but only 10-50 ng from HeLa cells. PLK-2 inhibition caused an increase in &#945;-synuclein mRNA in both the Input and BrU-IP (Figure 3B) suggesting that the increase in the steady state RNA is caused by an increase in synthesis of RNA. To ensure that PLK-2i does not just cause an overall increase in RNA production, 3 additional endogenous genes (ACTB, HMBS, and NADH) were measured (Figure 4). Neither of these genes was increased in the Input (Figure 4A) and BrU-IP (Figure 4B) upon PLK-2i treatment.
To validate the specificity of the BrU-IP, a control with non-BrU treated cells was included. The levels of 18sRNA captured by BrU-IP from BrU-treated and untreated cells were compared (Figure 5). This clearly demonstrates that the BrU-IP is highly specific and only captures a very small fraction of the non-labelled RNA (Figure 5).
Because the BrU-labelled RNA is only produced within 1 h, it is important to choose a proper reference gene, since some normally suitable genes might be expressed in too low concentrations to be reliable. 18sRNA was chosen as a reference gene for the data presented in Figure 3B and Figure 4 because of its high abundance. Several reference genes were, however, tested, and some showed high threshold cycles (&#62;30) (Figure 6). This data also demonstrates that the difference between BrU-labelled IP&#39;ed RNA and total RNA in the input is approximately 5 Ct&#39;s, and some RNAs expressed at low amounts in the cell might therefore not be properly detected following BrU-IP.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57056/57056fig1.jpg" /> 
Figure 1: Description of BrU-IP for investigation of RNA synthesis. (A) RNA is transcribed in cells from DNA by RNA polymerases. (B) Upon addition to the media, BrU is taken up by cells and incorporated into the newly transcribed RNA. (C) For measuring RNA synthesis rates: (1) newly synthesized RNA is labelled for 1 h with BrU, (2) RNA is extracted from the cells immediately after labelling, (3) BrU-labelled RNA is immunoprecipitated using anti-BrU antibodies, (4) BrU-labelled (and corresponding input) RNA is analyzed by RT-qPCR or sequencing. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57056/57056fig2.jpg" /> 
Figure 2: Assessment of labelling time. Example of an initial assessment of BrU-labelling time. AsPC-1 cells grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum and 50 U/mL / 50 &#181;g/mL penicillin/streptomycin were treated with BrU at a final concentration of 2 mM for 0, 1, 2, and 4.5 h before RNA extraction and analysis by RT-qPCR. Data is quantified as 2-Ct, and is presented as the mean of technical triplicates &#177; standard deviation, n = 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57056/57056fig3.jpg" /> 
Figure 3: Representative results showing Input and BrU-IP RNA from DMSO and PLK-2i treated cells. (A) 1 h and 4 h 2 mM BrU treatment did not induce any visible morphologic changes in HEK293T cells. Scale bar = 100 &#181;m. (B) Adapted figure from reference1. 3,500,000 HEK293T cells were seeded per 10-cm dish and transfected with a plasmid expressing &#945;-synuclein 24 h after seeding. 24 h post transfection cells were incubated for 1 h with BrU in the presence of DMSO or PLK-2i, followed by RNA extraction and BrU-IP. Analysis of RNA was done by RT-qPCR using probe sets directed against human SNCA (&#945;-synuclein) and 18sRNA. BrU-IP and Input SNCA was quantified relative to BrU-IP and Input 18sRNA, respectively, using the 2-&#916;&#916;CT method. Results were normalized to the DMSO-treated samples. Data is presented as mean &#177; standard deviations. Statistical comparisons were performed using Student&#39;s unpaired t-test, *p &#60;0.001, n = 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57056/57056fig4.jpg" /> 
Figure 4: Measurement of other genes not changed by PLK-2i. PLK-2i changes neither the total nor synthesized RNA of ACTB, HMBS, and NADH. (A) ACTB, HMBS, and NADH RNA from input were measured in triplicate in one experiment and quantified relative to 18sRNA in Input using the 2-&#916;&#916;CT method. Values were normalized to the DMSO treated samples and bars represent mean of triplicates &#177; standard deviations, n = 1. (B) ACTB, HMBS, and NADH were also measured in the BrU-IP&#39;ed RNA and quantified relative to 18sRNA in the BrU-IP. Values were normalized to DMSO and presented as in A, n = 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57056/57056fig5.jpg" /> 
Figure 5: Specificity of BrU-IP. For comparison of 18sRNA captured by BrU-IP from BrU- and non-BrU-treated cells, RNA from BrU-IP was validated relative to Input RNA to account for differences in starting material and hereafter normalized to BrU-treated samples. Only a very small fraction (0.002) of non-BrU-labelled 18sRNA was immunoprecipitated, demonstrating that the BrU-IP is highly specific. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57056/57056fig6.jpg" /> 
Figure 6: Levels of different mRNAs in BrU-IP RNA compared to Input RNA. Several potential reference genes were tested because some RNAs will be present in very low concentrations in BrU-IP RNA due to small amounts of produced RNA within the 1 h BrU treatment. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57056/57056fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

investigation-rna-synthesis-using-5-bromouridine-labelling

Nanyang Technological University

Research

JoVE Journal

Genetics

9.5K Views.  Aarhus University. Deze methode kan worden gebruikt voor het meten van RNA-synthese. 5-Bromouridine is toegevoegd aan de cellen en opgenomen in gesynthetiseerde RNA. RNA-synthese wordt gemeten door RNA extractie onmiddellijk na de etikettering, gevolgd door 5-Bromouridine-gerichte immunoprecipitation van gelabelde RNA en analyse door omgekeerde transcriptie en kwantitatieve polymerase-kettingreactie.

Video: Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Immunology and Infection

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Biochemistry

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biology

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

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