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 crosslinking of 4sU-labeled RNA to cellular proteins.
Start of labeling
<ol>
	<li>Thaw 4-thiouridine (4sU) just before use and pipette required amount of 4sU for each condition into a sterile Falcon tube.</li>
	<li>Take the required amount of cell culture medium (5 ml per 10 cm dish) off the dishes and add to 4sU-containing Falcon tube and mix thoroughly. Remove and discard the remaining medium from the dishes.</li>
	<li>Apply 4sU-containing medium back to the dishes.</li>
</ol>
End of labeling
<ol start="4">
	<li>Remove cell culture medium from cells. Add 5 ml of Trizol to each plate. For complex experiments including multiple time points or conditions, this step is best done by two people, one removing the medium, the other adding Trizol and harvesting the lysate.</li>
	<li>Incubate for 5 min at room temperature for complete cell lysis.</li>
	<li>Use a 10 ml pipette to rinse the plate carefully with the added Trizol. This aids complete cell lysis and sample recovery. Handle with care as Trizol is extremely hazardous when getting in contact with skin or eyes! Have antidote for phenol burns at hand (e.g. Polyethylene glycol 300 or 400 in industrial methylated spirits (70:30)). Transfer samples to polypropylene tubes. Please note that standard Falcon tubes do not resist these high g forces). Samples can be stored at -20 &deg;C for at least one month until total RNA is prepared.</li>
</ol>
2. RNA Preparation Using Modified Trizol Protocol
<ol>
	<li>Add 1 ml chloroform (0.2 ml per ml Trizol) and shake vigorously for 15 sec. Incubate at room temperature for 2 - 3 min.</li>
	<li>Centrifuge at 13,000 &times; g for 15 min at 4 &deg;C.</li>
	<li>Transfer aqueous upper phase (containing the RNA) to a new 15 ml polypropylene tube.</li>
	<li>Add &frac12; the reaction volume of both RNA precipitation buffer and isopropanol (e.g. to 3 ml supernatant add 1.5 ml RNA precipitation buffer and 1.5 ml isopropanol).</li>
	<li>Mix well. Incubate at room temperature for 10 min.</li>
	<li>Centrifuge at 13,000 &times; g for 10 min at 4 &deg;C. Discard supernatant.</li>
	<li>Spin down briefly (5,000 &times; g for 30 sec) and remove residual isopropanol with 200 &mu;l pipette.</li>
	<li>Add an equal volume of 75% ethanol and shake tube until the pellet detaches. Avoid breaking it into many small pieces as this may make removal of residual ethanol difficult.</li>
	<li>Centrifuge at 13,000 &times; g for 10 min at 4 &deg;C. Discard supernatant.</li>
	<li>Spin down RNA briefly and remove remaining ethanol with a 200 &mu;l pipette. Repeat step and remove remaining ethanol with a 20 &mu;l pipette. After these two steps, no further drying of the pellet should be performed.</li>
	<li>Add 100 &mu;l of H2O per 100 &mu;g expected RNA yield and mix well by pipetting up and down 5 - 6 times to aid in dissolving the RNA.</li>
	<li>Dissolve and denature RNA by heating to 65 &deg;C for 10 min (shaker) and immediately place on ice.</li>
	<li>Measure RNA concentration at 260 nm using a NanoDrop spectrophotometer, following manufacturer's instructions. This RNA can be stored at -80 &deg;C for at least one month.</li>
</ol>
3. Thiol-specific Biotinylation of Newly Transcribed RNA
<ol>
	<li>Start with 60 - 80 &mu;g of total cellular RNA.</li>
	<li>Constitute labeling reaction. Pipette in the following order (per &mu;g RNA):
	<ol class="lalpha">
		<li>1 &mu;l 10x Biotinylation Buffer</li>
		<li>7 &mu;l RNA (containing 1 &mu;g RNA diluted in nuclease-free H2O)</li>
		<li>2 &mu;l biotin-HPDP (1 mg/ml DMF)</li>
	</ol>
	</li>
</ol>
Always add the biotin-HPDP last and mix immediately by pipetting. In case the biotin precipitates, DMF content can be increased to a final concentration of 40%.
<ol start="3">
	<li>Incubate at room temperature for 1.5 hr with rotation.</li>
	<li>Add an equal volume of chloroform. Mix vigorously. Incubate for 2 - 3 minutes until the phases begin to separate and bubbles start to disappear.</li>
	<li>Centrifuge at 20,000 &times; g for 5 min at 4 &deg;C. Carefully transfer the upper aqueous phase into a new tube.</li>
	<li>Repeat steps 3.4 and 3.5 once. You may want to perform this step in 2 ml Phase Lock Gel Heavy tubes to reduce loss of RNA.</li>
	<li>RNA precipitation: add 1/10 the volume of 5 M NaCl and an equal volume of isopropanol to the water phase.</li>
	<li>Centrifuge at 20,000 &times; g for 20 min at 4 &deg;C. Discard supernatant.</li>
	<li>Add an equal volume of 75% ethanol, centrifuge at 20,000 &times; g for 10 min at 4 &deg;C, discard supernatant.</li>
	<li>Spin briefly and remove residual ethanol with 200 &mu;l pipette.</li>
	<li>Spin briefly and remove residual ethanol with 20 &mu;l pipette.</li>
	<li>Do not allow RNA to dry. Re-suspend it in 50 - 100 &mu;l H2O (~1 &mu;l per 1 &mu;g input RNA). Mix well by pipetting up and down 5 - 6 times.</li>
	<li>Check RNA quality by electrophoretical analysis to exclude RNA degradation.</li>
</ol>
4. Dot Blot Analysis of 4sU-incorporation (Optional)
4sU incorporation can be readily determined by dot blot analysis of biotinylated RNA. This is an optional step that allows trouble shooting and estimation of 4sU incorporation rates relative to a biotinylated DNA oligo control. For this assay we recommend using iodoacetyl-biotin instead of biotin-HPDP for biotinylation of 4sU-labeled RNA in step 3.2. This results in an irreversible biotinylation of 4sU-RNA. Therefore, column-based methods (e.g. RNeasy) can be used for recovery of much smaller amounts of biotinylated RNA (e.g. 5 &mu;g). While RNA biotinylated using biotin-HPDP is also suitable for this assay, the resulting signal is weaker and the signal-noise ratio less favorable (Figure 3).
<ol>
	<li>Follow the protocol for 4sU-labeling and isolation of total cellular RNA as described in sections 1 and 2.</li>
	<li>Biotinylate 4sU-labeled RNA as described in section 3 replacing biotin-HPDP with iodoacetyl-biotin and perform two chloroform extractions to completely remove excessive iodoacetyl-biotin residues.</li>
	<li>Recover biotinylated RNA by isopropanol/ethanol precipitation as described or using a column-based approach (e.g. RNeasy) in case small amounts of RNA (&lt;10 &mu;g) are used.</li>
	<li>Incubate the Zeta membrane in nuclease-free water with rocking for 10 min.</li>
	<li>Take the membrane out of the nuclease-free water and remove excessive fluids by placing membrane in between two clean paper towels and pressing firmly. Air-drying the membrane for 5 min will result in nicer dots.</li>
	<li>For each sample, prepare 20 &mu;l of 200 ng/&mu;l RNA using ice cold dot blot binding buffer (10 mM NaOH, 1 mM EDTA). Apply 5 &mu;l of this dilution (i.e. 1 &mu;g of RNA) as well as three subsequent 10-fold dilutions (i.e. 100, 10, and 1 ng RNA, respectively) to the Zeta membrane by pipetting. Pipetting through an empty rack of pipette tips can be employed to provide evenly distributed spacing. Alternatively, use a dot blot apparatus according to the manufacturer's instructions.</li>
	<li>Apply 5 &mu;l of the biotin-labeled DNA oligo at concentrations ranging from 20 ng/&mu;l to 20 pg/&mu;l (i.e. 100 to 0.1 ng oligo) as a positive control to the membrane by pipetting. Use a biotinylated, 4sU-naive sample as negative control.</li>
	<li>Air-dry the membrane for 5 min.</li>
	<li>Incubate the membrane for 30 min in 40 ml blocking buffer with rocking.</li>
	<li>Incubate the membrane with 10 ml of 1:1,000 streptavidin-horseradish peroxidase for 15 min (5 ml PBS + 5 ml 20% SDS + 10 &mu;l streptavidin-horseradish peroxidase)</li>
	<li>Wash membrane twice in 40 ml PBS + 10% SDS (20 ml PBS + 20 ml 20% SDS) for 5 min.</li>
	<li>Wash membrane twice in 40 ml PBS + 1% SDS (38 ml PBS + 2 ml 20% SDS) for 5 min.</li>
	<li>Wash membrane twice in 40 ml PBS + 0.1% SDS (40 ml PBS + 200 &mu;l 20% SDS) for 5 min.</li>
	<li>Remove excessive fluid by placing membrane in between two clean paper towels and pressing on them firmly.</li>
	<li>Visualize membrane-bound HRP using ECL per manufacturer's instructions.</li>
	<li>Place the membrane in plastic foil/bag, remove air bubbles and incubate for 2 min in the dark.</li>
	<li>Expose membrane to film for 1 - 5 min.</li>
</ol>
5. Separation of Labeled and Unlabeled RNA Using Streptavidin-coated Magnetic Beads
<ol>
	<li>Heat washing buffer (3 ml per sample) to 65 &deg;C in a water bath.</li>
	<li>Prepare fresh 100 mM dithiothreitol (DTT) in nuclease-free H2O. Do so by decanting 15 - 30 mg of DTT powder into a clean 50 ml Falcon tube placed on the ultra-fine scale. Weigh and add required amount of nuclease-free H2O.</li>
	<li>Heat biotinylated RNA samples to 65 &deg;C for 10 min to denature and immediately place on ice.</li>
	<li>Place &mu;Macs columns into the magnetic stand. We recommend not to process more than 12 samples at a time (6 - 8 samples are optimal).</li>
	<li>Pre-equilibrate Miltenyi columns with 1 ml room temperature washing buffer. This will take about 15 min.</li>
	<li>Meanwhile, add 100 &mu;l of streptavidin beads to 50 - 100 &mu;l of biotinylated RNA. Incubate at room temperature for 15 min with rotation.</li>
	<li>If any of the columns has not initiated draining by now this can be facilitated by gently pressing on the top of the column with a gloved finger. Once the flow has started the columns drain readily.</li>
	<li>Apply the RNA/beads to the columns. Discard the flow-through unless you want to recover the unlabeled RNA fraction (see section 7).</li>
	<li>Wash three times with 0.9 ml of 65 &deg;C washing buffer (1 ml pipette tips shrink when pipetting buffers at 65 &deg;C).</li>
	<li>Wash three times with 0.9 ml room temperature washing buffer.</li>
	<li>Pipette 700 &mu;l Buffer RLT (RNeasy MinElute Cleanup Kit, Qiagen) into new 2 ml tubes and place them underneath the columns.</li>
	<li>Elute the newly transcribed RNA into the RLT Buffer by adding 100 &mu;l of 100 mM DTT to the columns.</li>
	<li>Perform a second elution round 3 min later into the same tube by adding another 100 &mu;l of 100 mM DTT.</li>
</ol>
6. Recovery of Newly Transcribed RNA
Continue with the RNeasy MinElute Cleanup (Qiagen) protocol following the manufacturer's instructions. Elute in 25 &mu;l nuclease-free H2O. Measure RNA concentrations using a Nanodrop Spectrophotometer. To avoid the need to thaw and re-freeze RNA before submitting it to a high-throughput assay, we recommend preparing cDNA immediately after the newly transcribed RNA is purified. Use 2.5 &mu;l of the newly transcribed RNA in 20 &mu;l cDNA synthesis mix for cDNA synthesis following the manufacturer's instructions. Perform qRT-PCR controls using 1:10 dilutions of the cDNA mix. Store RNA at -80 &deg;C.
7. Recovery of Unlabeled, Unbound RNA (Optional)
In case the unbound RNA needs to be recovered; collect and combine the flow-through (after adding the RNA-streptavidin beads solution to the columns) and the first wash for subsequent precipitation. Usually it is sufficient to precipitate only 50% of the unbound RNA as this will contain &gt;80% of the starting material.
<ol>
	<li>Add an equal volume of isopropanol (no salt needs to be added as the washing buffer already contains 1 M NaCl).</li>
	<li>Centrifuge at 20,000 &times; g for 20 min at 4 &deg;C. Discard supernatant.</li>
	<li>Add an equal volume of 75% ethanol, centrifuge at 20,000 &times; g for 10 min at 4 &deg;C, discard supernatant.</li>
	<li>Spin briefly and remove residual ethanol with 200 &mu;l pipette.</li>
	<li>Spin briefly and remove residual ethanol with 20 &mu;l pipette.</li>
	<li>Do not allow RNA to dry. Resuspend it in 100 &mu;l H2O. Mix well by pipetting up and down 5 - 6 times. Incubate at 65 &deg;C for 10 min with shaking and transfer directly to ice.</li>
	<li>Check RNA quality by electrophoretical analysis to exclude RNA degradation.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 ...

Watch this Scientific Journal Video about Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture at JoVE.com

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 ...

metabolic-labeling-newly-transcribed-rna-for-high-resolution-gene

Research

JoVE Journal

Biology

27.0K Views.  Max von Pettenkofer Institute. 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.

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

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 ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...