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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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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T., 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=Use+of+Bru-Seq+and+BruChase-Seq+for+genome-wide+assessment+of+the+synthesis+and+stability+of+RNA.">Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.</a> Methods. 67, 45-54 (2014).</li><li>D&#246;lken, L., 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=High-resolution+gene+expression+profiling+for+simultaneous+kinetic+parameter+analysis+of+RNA+synthesis+and+decay.">High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay.</a> Rna. , 1959-1972 (2008).</li><li>Raghavan, A., 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=Genome-wide+analysis+of+mRNA+decay+in+resting+and+activated+primary+human+T+lymphocytes.">Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes.</a> Nucleic Acids Res. 30, 5529-5538 (2002).</li><li>Sharova, L. V., 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=Database+for+mRNA+half-life+of+19+977+genes+obtained+by+DNA+microarray+analysis+of+pluripotent+and+differentiating+mouse+embryonic+stem+cells.">Database for mRNA half-life of 19 977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells.</a> DNA Res. 16, 45-58 (2009).</li><li>Pandya-Jones, A., 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=Splicing+kinetics+and+transcript+release+from+the+chromatin+compartment+limit+the+rate+of+Lipid+A-induced+gene+expression.">Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression.</a> RNA. 19, 811-827 (2013).</li></ol>

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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Genetics

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.