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><tbody><tr><td>4-thiouridine</td><td>Carbosynth</td><td>T4509</td><td>Prepare 50 mM stock in sterile H&lt;sub&gt;2&lt;/sub&gt;O, store at -20 &amp;deg;C in aliquots of 50-500 &amp;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>&lt;strong&gt;WARNING - CORROSIVE and HAZARDOUS TO HEALTH!&lt;/strong&gt; Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 &amp;deg;C.</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>372978</td><td>&lt;strong&gt;WARNING - HAZARDOUS TO HEALTH&lt;/strong&gt;</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>650447</td><td>&lt;em&gt;&lt;/em&gt;</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 H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;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 &amp;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&#039;-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 &amp;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 &amp;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 &amp;deg;C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 &amp;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&#039;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>&amp;mu;Macs Streptavidin Kit</td><td>Miltenyi</td><td>130-074-101</td><td>Store the beads at 4 &amp;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 H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>43817</td><td>Prepare as 100 mM DTT in nuclease-free H&lt;sub&gt;2&lt;/sub&gt;O, always prepare fresh before use.</td></tr><tr><td>RNeasy MinElute Kit</td><td>Qiagen</td><td>74204</td><td>Store columns at 4 &amp;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 colspan="4">All solutions/reagents should be stored at room temperature unless otherwise specified.</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt; </td></tr><tr><td>UV/VIS spectrophotometer</td><td>Thermo Scientific</td><td>NanoDrop 1000</td><td>Or equivalent. Use low volume (1-2 &amp;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 &amp;#215; 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&amp;#215;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&amp;#215;g</td></tr><tr><td>Adaptors for 15 ml tubes</td><td>Laborger&amp;#228;te Beranek</td><td>356964</td><td>Or equivalent equipment capable of reaching 13,000&amp;#215;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 &amp;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;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 μg of total RNA. Starting with less than 30 μg of total RNA results in small RNA pellets that are hard to see after the biotinylation step and thus may be easi...

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 ...

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Research

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Biology

27.1K 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

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

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

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

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

Genetics

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

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

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

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

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

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

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 ...

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

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

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

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

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

Biochemistry

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
</div>
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In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
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Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
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You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

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

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