We would like to thank Amie Regan for careful reading of the manuscript. This work was supported by NGFN Plus grant #01GS0801, MRC fellowship grant G1002523 and NHSBT grant WP11-05 to L.D. and DFG grant FR2938/1-1 to C.C.F.

1. Metabolic Labeling with 4-thiouridine
Make a detailed plan of the experimental setup/schedule, e.g. when to add the 4sU to cell culture and when to harvest the samples. Plan for at least 5 min in between each condition. Only treat cells of one condition at a time. Handle max. 3 - 5 dishes at a given time. Handle cells as quickly as possible to minimize changes in temperature and CO2 levels. Avoid exposing the cells to bright light after 4sU is added as this may result in c...

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Metabolic labeling of newly transcribed RNA substantially enhances the power of high-throughput technologies like microarrays and RNA-seq by providing more suitable templates to address the biological question of interest. The present protocol underwent extensive optimization. It allows &gt;1,000-fold enrichment of newly transcribed RNA and provides highly reproducible results.
The experimental design of a 4sU-tagging experiment is of crucial importance as newly transcribed RNA will depi...

Gene expression profiling is a key tool used to study cellular processes and the associated complex interaction network. Studies on mRNA abundance have typically been the method of choice to obtain basic insights into the underlying molecular mechanisms. The development of whole-transcriptome microarrays 1 and, more recently, next-generation sequencing of RNA (RNA-seq) 2-4 fueled this approach. While these technologies have revolutionized our understanding of the complexity of cellular gene expression, they face major limitations due to intrinsic properties of their template sample, i.e. total cellular RNA. First, short-term changes in total RNA levels do not match changes in transcription rates, but are inherently dependent on the RNA half-life of the respective transcripts. While a fivefold induction of a short-lived transcript, e.g. encoding for a transcription factor, will be readily detectable in total RNA within an hour, the same induction of a long-lived transcript, e.g. encoding for a metabolic enzyme, will remain virtually invisible. In addition, even a complete shut-down (&gt;1,000-fold down-regulation) in the transcription rate of an average gene with an RNA half-life of five hours will simply take five hours for its total RNA levels to decrease by only twofold. Therefore, analysis of total RNA favors the detection of up-regulation of short-lived transcripts, many of which encode for transcription factors and genes with regulatory functions 5. In addition, the true kinetic cascade of regulation is obscured and primary signaling events cannot be differentiated from secondary. Both, in turn, may result in substantial bias in downstream bioinformatics analyses. Second, alterations in total RNA levels cannot be attributed to changes in RNA synthesis or decay. Measurements of the latter require cell invasive approaches, e.g. blocking transcription using actinomycin D 6, and extended monitoring of ongoing RNA decay over time. With a mean mRNA half-life in mammalian cells of 5 - 10 hr 5,7, mRNA levels of most genes will only have decreased by less than twofold following several hours of transcriptional arrest. These rather small differences result in grossly imprecise measurements of mRNA half-lives for the majority of cellular genes due to the exponential nature of the underlying mathematical equations. Finally, while RNA-seq of total cellular RNA revealed that approximately half of our genes are subject to alternative splicing events8, the underlying kinetics as well as the dynamic mechanisms guiding tissue- and context-specific regulation of RNA processing remain poorly understood. In addition, the contribution of RNA processing to differential gene expression, particularly for non-coding RNAs, remains to be determined. Altogether, these limitations represent major obstacles for bioinformatic kinetic modeling of the underlying molecular mechanisms.
We recently developed an approach, termed high resolution gene expression profiling, to overcome these problems 5,7,9. It is based on metabolic labeling of newly transcribed RNA using 4-thiouridine (4sU-tagging), a naturally occurring uridine derivative, and provides direct access to newly transcribed transcripts with minimal interference in cell growth and gene expression (see Figure 1) 5,10-12. Exposure of eukaryotic cells to 4sU results in its rapid uptake, phosphorylation to 4sU-triphosphate, and incorporation into newly transcribed RNA. Following isolation of total cellular RNA, the 4sU-labeled RNA fraction is thiol-specifically biotinylated generating a disulfide bond between biotin and the newly transcribed RNA. 'Total cellular RNA' can then be quantitatively separated into labeled ('newly transcribed') and unlabeled ('pre-existing') RNA with high purity using streptavidin-coated magnetic beads. Finally, labeled RNA is recovered from the beads by simply adding a reducing agent (e.g. dithiothreitol) cleaving the disulfide bond and releasing the newly transcribed RNA from the beads.
Newly transcribed RNA depicts the transcriptional activity of every gene during the timeframe of 4sU exposure. 4sU-tagging in the timescale of minutes thus provides a snapshot picture of eukaryotic gene expression and an ideal template for down-stream bioinformatic analyses (e.g. promoter analysis). In cases where steady-state conditions can be assumed, the ratios of newly transcribed/total, newly transcribed/unlabeled and unlabeled/total RNA provide non-invasive access to precise RNA half-lives 7,13. In addition, it is important to note that newly transcribed RNA purified after as little as 5 min of 4sU-tagging (5 min 4sU-RNA) is younger than 15 and 60 min 4sU-RNA. When performing both ultra-short and progressively longer 4sU-tagging in a single experimental setting combined with RNA-seq, the kinetics of RNA processing are revealed at nucleotide resolution 9. Finally, time-course analyses of newly transcribed and total RNA combined with computational modeling allow an integrative analysis of RNA synthesis and decay 14.
In conclusion, this approach allows for the direct analysis of the dynamics of RNA synthesis, processing, and degradation in eukaryotic cells. It is applicable in all major model organisms including mammals, insects (Drosophila), amphibians (Xenopus), and yeast 5,15,16. It is directly compatible with microarray analysis 5,17, RNA-seq 9,13,14, and is applicable in vivo12,15. Here, we detail the methodology to label, isolate, and purify newly transcribed RNA in cultured mammalian cells. In addition, potential problems and pitfalls are discussed.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>4-thiouridine</td>
 <td>Carbosynth</td>
 <td>T4509</td>
 <td>Prepare 50 mM stock in sterile H2O, store at -20 &deg;C in aliquots of 50-500 &mu;l, discard unused reagent, do not refreeze.</td>
 </tr>
 <tr>
 <td>Trizol</td>
 <td>Invitrogen</td>
 <td>15596026 (100 ml), 15596018 (200 ml)</td>
 <td>WARNING - CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma</td>
 <td>372978</td>
 <td>WARNING - HAZARDOUS TO HEALTH</td>
 </tr>
 <tr>
 <td>Isopropanol</td>
 <td>Sigma</td>
 <td>650447</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium citrate, nuclease-free</td>
 <td>Sigma</td>
 <td>C8532</td>
 <td>Prepare 1.6 M stock solution using nuclease-free water.</td>
 </tr>
 <tr>
 <td>5M nuclease-free NaCl</td>
 <td>Sigma</td>
 <td>71386</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>Nuclease-free H2O</td>
 <td>Sigma</td>
 <td>W4502</td>
 <td>Make 1 ml aliquots in nuclease-free tubes.</td>
 </tr>
 <tr>
 <td>RNA precipitation buffer</td>
 <td></td>
 <td></td>
 <td>1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under strictly nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma</td>
 <td>459844</td>
 <td>Use with nuclease-free water to prepare 80% ethanol, store at -20 &deg;C.</td>
 </tr>
 <tr>
 <td>1 M nuclease-free Tris Cl, pH 7.5</td>
 <td>Lonza</td>
 <td>51237</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>500 mM nuclease-free EDTA, pH 8.0</td>
 <td>Invitrogen</td>
 <td>15575-020</td>
 <td>Stock solution</td>
 </tr>
 <tr>
 <td>10x Biotinylation Buffer (BB)</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water, make aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Dimethylformamide (DMF)</td>
 <td>Sigma</td>
 <td>D4551</td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-Link biotin-HPDP</td>
 <td>Pierce</td>
 <td>21341</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml.</td>
 </tr>
 <tr>
 <td>Phase Lock Gel Heavy tubes 2.0 ml</td>
 <td>Eppendorf</td>
 <td>0032 005.152</td>
 <td>Optional for the chloroform extraction step.</td>
 </tr>
 <tr>
 <td>Zeta membrane</td>
 <td>BIORAD</td>
 <td>162-0153</td>
 <td></td>
 </tr>
 <tr>
 <td>10x Dot blot binding buffer</td>
 <td></td>
 <td></td>
 <td>100 mM NaOH, 10 mM EDTA</td>
 </tr>
 <tr>
 <td>Biotin-oligo</td>
 <td></td>
 <td></td>
 <td>5'-biotin, 25 nucleotides, any sequence</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulphate</td>
 <td>Fisher</td>
 <td>BPE9738</td>
 <td>For 100 ml 20% stock solution, add 20 g SDS to 80 ml PBS pH 7-8 and adjust volume to 100 ml. Keep all high-percentage SDS solutions above 20 &deg;C. Warm the solutions slightly should SDS precipitate.</td>
 </tr>
 <tr>
 <td>EZ-Link Iodoacetyl-LC-Biotin</td>
 <td>Pierce</td>
 <td>21333</td>
 <td>Prepare 1 mg/ml stock solution by dissolving 50 mg iodoacetyl-biotin in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 &deg;C in aliquots of 1 ml. Generates irreversible, thiol-specific biotinylation.</td>
 </tr>
 <tr>
 <td>Phosphate buffer saline</td>
 <td>Gibco</td>
 <td>10010-015</td>
 <td></td>
 </tr>
 <tr>
 <td>Dot blot blocking buffer</td>
 <td></td>
 <td></td>
 <td>Mix 20 ml 20% SDS with 20 ml 1 x PBS pH 7-8 and add EDTA to the final concentration of 1 mM.</td>
 </tr>
 <tr>
 <td>Streptavidin-horseradish peroxidase </td>
 <td>Vector Laboratories</td>
 <td>SA5004</td>
 <td>Store at -20 &deg;C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 &mu;l Streptavidin-HRP before use. </td>
 </tr>
 <tr>
 <td>ECL reagent</td>
 <td>GE Healthcare</td>
 <td>RNP2109</td>
 <td>Use following the manufacturer's instructions.</td>
 </tr>
 <tr>
 <td>Super RX, X-RA Film, 18x24 cm</td>
 <td>Fujifilm</td>
 <td>47410 19236</td>
 <td></td>
 </tr>
 <tr>
 <td>&mu;Macs Streptavidin Kit</td>
 <td>Miltenyi</td>
 <td>130-074-101</td>
 <td>Store the beads at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P1379</td>
 <td></td>
 </tr>
 <tr>
 <td>Washing buffer</td>
 <td></td>
 <td></td>
 <td>100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O.</td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>Sigma</td>
 <td>43817</td>
 <td>Prepare as 100 mM DTT in nuclease-free H2O, always prepare fresh before use.</td>
 </tr>
 <tr>
 <td>RNeasy MinElute Kit</td>
 <td>Qiagen</td>
 <td>74204</td>
 <td>Store columns at 4 &deg;C, remaining components of the kit at room temperature.</td>
 </tr>
 <tr>
 <td>1.5 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.692.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>2.0 ml screw-top polypropylene tubes</td>
 <td>Sarstedt</td>
 <td>72.694.005</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td>50 ml tubes</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>Compatible with Dimethylformamide</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>All solutions/reagents should be stored at room temperature unless otherwise specified.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>UV/VIS spectrophotometer</td>
 <td>Thermo Scientific</td>
 <td>NanoDrop 1000</td>
 <td>Or equivalent. Use low volume (1-2 &mu;l) for measurements of low RNA concentrations to avoid excessive sample loss.</td>
 </tr>
 <tr>
 <td>Polypropylene 15 ml centrifuge tubes</td>
 <td>VWR International</td>
 <td>525-0153</td>
 <td>In contrast to standard 15 ml tubes, these tolerate up to 15,000 &times; g</td>
 </tr>
 <tr>
 <td>High-speed centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-25</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>High-speed rotor</td>
 <td>Beckman Coulter</td>
 <td>JLA-16250</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Adaptors for 15 ml tubes</td>
 <td>Laborger&auml;te Beranek</td>
 <td>356964</td>
 <td>Or equivalent equipment capable of reaching 13,000&times;g</td>
 </tr>
 <tr>
 <td>Refrigerated table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5430 R</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Thermomixer</td>
 <td>Eppendorf</td>
 <td>Thermomixer compact</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Magnetic stand</td>
 <td>Miltenyi Biotec</td>
 <td>130-042-109</td>
 <td>One stand holds 8 &mu;Macs columns.</td>
 </tr>
 <tr>
 <td>Waterbath</td>
 <td>Grant</td>
 <td>SUB Aqua 5</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>Ultra-fine scale</td>
 <td>A&amp;D</td>
 <td>GR-202</td>
 <td>Or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel iBase Power System</td>
 <td>Invitrogen</td>
 <td>G6400UK</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 <tr>
 <td>E-Gel EX 1% agarose precast gels</td>
 <td>Invitrogen</td>
 <td>G4020-01</td>
 <td>For RNA gels; or equivalent.</td>
 </tr>
 </tbody>
</table>

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.

1. Starting Material and Expected Yields
Following 1 hour (hr) of 4sU-exposure newly transcribed RNA represents about 1 - 4% of total cellular RNA. This will be lower in growth-arrested cells as they no longer synthesize RNA to account for cell growth/replication. When labeling for 1 hr, we recommend starting the assay with 60 - 80 &mu;g of total RNA. Starting with less than 30 &mu;g of total RNA results in small RNA pellets that are hard to see after the biotinylation step and thus may be ...

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

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 ...

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Research

JoVE Journal

Biology

26.9K 조회수.  Max von Pettenkofer Institute. 전체 세포 RN​​A 단기 RNA 합성과 붕괴의 변화뿐만 아니라 RNA 처리 속도론을 연구 가난한 템플릿을 제공합니다. 여기에, 우리는 4 thiouridine에 새로 전사 RNA의 대사 라벨을 설명하는 이러한 한계를 극복 할 수 있도록 새로 전사 RNA의 싸이 특정 biotinylation 및 정제에 의해 따랐다.