Name: determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts
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HAC was the Milton E. Cassel scholar of the Rita Allen Foundation (http://www.ritaallenfoundation.org). This work was funded by grants to HAC from the National Institute of General Medical Sciences Center of Excellence grant P50 GM071508 (P.I. David Botstein), PhRMA Foundation grant 2007RSGl9572, National Science Foundation Grant OCI-1047879 to David August, National Institute of General Medical Sciences R01 GM081686, National Institute of General Medical Sciences R01 GM0866465, the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research, the Iris Cantor Women&#8217;s Health Center/UCLA CTSI NIH Grant UL1TR000124, and the Leukemia Lymphoma Society. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number P50CA092131. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HAC is a member of the Eli &#38; Edythe Broad Center of Regenerative Medicine &#38; Stem Cell Research, the UCLA Molecular Biology Institute, and the UCLA Bioinformatics Interdepartmental Program.

All experiments described were approved by Institutional Review Boards at Princeton University and the University of California, Los Angeles.
1. Prepare Proliferating and Contact-inhibited Fibroblasts for ActD Time Course
NOTE: This protocol uses a timecourse with four timepoints. Three biologically independent samples can be collected per timepoint by collecting different tissue culture plates in one experiment, or the experiment can be repeated multiple times with d...

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Quiescence can be induced by external signals including withdrawal of mitogens or serum, lack of cell adhesion, and contact inhibition. Contact inhibition, one of multiple possible methods for inducing quiescence, is a highly evolutionarily conserved process in which cells exit the proliferative cell cycle in response to cell-to-cell contact. We focus here on contact inhibition as an example of a method to induce quiescence. Previous studies have reported that cell-cell contact can affect microRNA biogenesis<sup class="x...

Steady state levels of transcripts reflect the contribution of both transcript synthesis and transcript decay. Regulated and coordinated transcript decay is an important mechanism for controlling biological processes1,2,3,4. For example, transcript decay rates have been shown to contribute to the temporal series of events following activation by the inflammatory cytokine tumor necrosis factor5.
We have previously shown that the transition between proliferation and quiescence in primary human fibroblasts is associated with changes in the transcript levels of many genes6. Some of these changes reflect differences in the activity of transcription factors between these two states.
Changes in a transcript&#39;s decay rate can also contribute to changes in the expression level of a transcript in two different states7,8. Based on our earlier findings that the transition between proliferation and quiescence is associated with changes in the levels of multiple microRNAs9, we asked whether there is also a contribution from differential transcript decay in the changes in gene expression in proliferating versus quiescent fibroblasts.
In order to monitor transcript stability, we determined the rate of decay of transcripts genome-wide in proliferating versus quiescent cells. To achieve this, we monitored decay rates in proliferating versus quiescent fibroblasts by introducing an inhibitor of new transcription and monitoring the rate at which individual transcripts disappeared over time. The advantage of this approach is that, as compared to methods that simply monitor overall gene expression, by inhibiting new transcript synthesis, we will be able to determine the decay rate for these transcripts separately from the rate at which they are transcribed.
ActD treatment to inhibit new transcription and determine transcript decay rates has been successfully applied in multiple previous studies. ActD has been used to dissect the importance of RNA stability in the changes in transcript abundance that result from treatment with pro-inflammatory cytokines5. A similar approach has also been used to dissect the differences in transcript decay rate in proliferating versus differentiated C2C12 cells as they adopt a differentiated muscle phenotype10. As another example, global mRNA half-lives have also been detected in pluripotent and differentiated mouse embryonic stem cells11. In these examples, mRNA decay has been shown to be important for regulating transcript abundance and for the transition of cells to different cell states.
Applying the methods described below, we discovered changes in transcript decay rates in approximately 500 genes when comparing fibroblasts in proliferating and quiescent states12. In particular, we discovered that the targets of the microRNA miR-29, which is downregulated in quiescent cells, are stabilized when cells transition to quiescence. We describe here the methodology we used to determine decay rates in proliferating and quiescent cells. This methodology is useful for comparing global mRNA decay rates in two distinct but similar conditions when information about rapidly decaying genes is sought. It could also be used to address other questions such as the effect of cell culture conditions on transcript decay, for instance, in two dimensional versus three dimensional cultures. Decay rates can be determined genome-wide with methods such as microarrays or RNA-Seq. Alternatively, real-time qPCR or Northern blotting can be used to determine decay rates on a gene-by-gene or isoform-by-isoform basis. These rates can then be used to calculate the half-life of each monitored gene and to identify genes with decay rates that are different in two conditions.

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			<td height="42" style="height:42px;width:291px;">Centrifuges for microcentrifuge tubes capable of reaching 12,000 x g and 4&#176;C</td>
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			<td height="21" style="height:21px;width:291px;">Equipment for running agarose gels</td>
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			<td height="42" style="height:42px;width:291px;">Sterile tissue culture plates and conical tubes</td>
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			<td height="21" style="height:21px;width:291px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">11965-118</td>
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			<td height="21" style="height:21px;width:291px;">Fetal bovine serum</td>
			<td style="width:163px;">VWR</td>
			<td style="width:119px;">35-010-CV</td>
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			<td height="42" style="height:42px;width:291px;">Sterile serological pipets, pipettors and pipet tips for tissue culture</td>
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			<td height="63" style="height:63px;width:291px;">Individually wrapped, disposable Rnase-free pipettes, pipette tips and tubes for RNA isolation and analysis</td>
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			<td height="42" style="height:42px;width:291px;">Disposable gloves to be worn when handling reagents and RNA samples</td>
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			<td height="42" style="height:42px;width:291px;">RNaseZap</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">AM9780</td>
			<td style="width:237px;">For decontaminating work surfaces from RNase</td>
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			<td height="21" style="height:21px;width:291px;">2.0 ml eppendorf tubes</td>
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			<td height="21" style="height:21px;width:291px;">Trypsin-EDTA&#160; Solution 10X</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">9002-07-07</td>
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			<td height="22" style="height:23px;width:291px;">Sterile PBS</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
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			<td height="42" style="height:42px;width:291px;">Trizol</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15596018</td>
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			<td height="21" style="height:21px;width:291px;">Actinomycin D</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">A1410-2 mg</td>
			<td style="width:237px;">inhibits transcription</td>
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			<td height="21" style="height:21px;width:291px;">Sterile DMSO</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">31-761-00ML</td>
			<td style="width:237px;">solvent for actinomycin D</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Chloroform</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN19400225</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Isopropanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP2618500</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethanol</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP28184</td>
			<td style="width:237px;">molecular biology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">RNase-free Glycogen (20 mg/ml aqueous solution)</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0551</td>
			<td style="width:237px;">carrier for RNA precipitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:291px;">TURBO DNA-free Kit</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1907</td>
			<td style="width:237px;">removes DNA with DNase, then, in a subsequent step,&#160; inactivates DNA and removes divalent cations</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Agarose</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ICN820721</td>
			<td style="width:237px;">MP Biomedicals, Inc product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Loading dye for RNA gel</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0641</td>
			<td style="width:237px;">suitable even for denaturing electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Millenium RNA markers</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7150</td>
			<td style="width:237px;">RNA ladder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">One-color RNA Spike-in Kit</td>
			<td style="width:163px;">Agilent Technology</td>
			<td style="width:119px;">5188-5282</td>
			<td style="width:237px;">Example spike-in control for Agient microarray analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Tris base</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP152-1</td>
			<td style="width:237px;">molecular biology grade, for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Glacial acetic acid</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">A38-500</td>
			<td style="width:237px;">for TAE buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">EDTA</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td style="width:237px;">electrophoresis grade, for gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ethidium bromide</td>
			<td style="width:163px;">Millipore Sigma</td>
			<td style="width:119px;">E7637</td>
			<td style="width:237px;">for molecular biology</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">External RNA Controls Consortium RNA Spike-in Mix</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td>4456740</td>
			<td style="width:237px;">Spike-in control for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">TruSeq Stranded mRNA Library Preparation Kit A (48 samples, 12 indexes)</td>
			<td style="width:163px;">Illumina</td>
			<td style="width:119px;">RS-122-2101</td>
			<td style="width:237px;">for RNA Seq</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">96-well 0.3 ml PCR plate</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AB-0600</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Microseal B adhesive seals</td>
			<td style="width:163px;">Bio-Rad</td>
			<td style="width:119px;">MSB1001</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Rnase/Dnase-free Reagent Reservoirs</td>
			<td style="width:163px;">VWR</td>
			<td style="width:119px;">89094-662</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Rnase/Dnase-free Eight tube strips and caps</td>
			<td style="width:163px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM12230</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">SuperScript Reverse Transcriptase</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">18090010</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">AMPure XP Beads</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:237px;">for real-time qPCR</td>
		</tr>
	</tbody>
</table>

determining genome-wide transcript decay rates in proliferating and quiescent human fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We have previously reported the results of microarray analyses of transcript decay rates in proliferating and contact-inhibited primary human fibroblasts over an 8-hour time course12. A list of genes with a significant change in transcript stability comparing proliferating and contact-inhibited fibroblasts is provided in Supplementary Table 1. The fluorescence intensities at time zero and over a time course after ActD treatment are provided. Genes ...

Watch this Scientific Journal Video about Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts at JoVE.com

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

The overall goal of this procedure is to monitor transcript decay rates in proliferating and quiescent fibroblasts. This method can help answer key questions in the molecular biology field such as whether changes in gene expression result from changes in transcript decay rate. The main advantage of this technique is that it provides information on transcript decay rate, not just transcript abundance.To begin the protocol, establish proliferating and quiescent cultures of fibroblasts. Proliferating fibroblasts should be trypsinized regularly. Quiescent cultures can be established by allowing plates to reach confluence with regular medium changes.Add ActD 150 microliters to prewarmed medium 10 milliliters for all biological replicate cell culture plates. Add ActD at a concentration of 15 micrograms per milliliter of medium. Mix the solution well.Aspirate the old medium, and add medium containing ActD to the cells. To collect the zero time point sample, gently aspirate off the ActD medium and pipette between five and 10 milliliters of prewarmed PBS onto a 100-millimeter plate, five milliliters of solution for a 100-millimeter plate, two milliliters for a well of a six-well plate, and one milliliter for a well of a 12-well plate. Gently aspirate the PBS.Then add phenol-guanidine isothiocyanate solution directly to the tissue culture plate. Pipette the phenol-guanidine isothiocyanate solution cell mixture up and down several times to make a homogenous mixture. Collect each time point after the appropriate amount of time has passed, using the method described earlier.Incubate the samples at room temperature for five minutes to ensure the complete dissolution of RNA protein complexes. Next introduce spike-in controlled transcripts that can be detected with the methodology used. Introduce these controls into each sample at a known quantity and concentration.Using a pipette, transfer the phenylguanidine isothiocyanate solution mixture to a 2.0-milliliter microcentrifuge tube. Then add 0.2 milliliters chloroform to the phenylguanidine isothiocyanate solution mixture for every one milliliter of phenylguanidine isothiocyanate solution used. Shake the microcentrifuge tube vigorously for 15 seconds and then incubate the chloroform phenylguanidine isothiocyanate.After incubation, spin the samples at 12, 000 times g for 20 minutes at four degrees Celsius. The sample should separate into three layers. Transfer the aqueous layer to a new 2.0-milliliter microcentrifuge tube using a micropipette and be careful not to disturb the interphase during this process.Discard the interphase and organic fractions. Then add an equal volume of 2-Propanol to the aqueous fraction and invert the tube 10 times. Add up to one microliter of 20 milligram per milliliter aqueous glycogen per 20 microliters of sample, especially if the yield is expected to be low.Incubate the samples at room temperature for 10 minutes. After incubation, spin the samples. Ensure that at the end of the spin, the RNA has precipitated to form a while pellet at the bottom of the tube.With a pipette, remove and discard the supernatant, taking special care not to disturb the white RNA pellet. Add one milliliter of 75%ethanol per one milliliter of phenylguanidine isothiocyanate solution reagent to wash the pellet. Spin the samples at 12, 000 times g for 10 minutes.After removing most of the supernatant, use a pipette to remove as much ethanol as possible but do not disturb the pellet. Spins the samples to pellet all excess ethanol. The RNA pellet is less compact at this step and tends to move.After the spin, remove all excess ethanol from the tube without disturbing the pellet. Allow the RNA pellet to air-dry for five minutes. Add 50 microliters of nuclease-free water to the RNA pellet and resuspend the pellet in nuclease-free water.Treat the samples of one microliter Deanase to remove DNA and then inactivate the Deanase and cations. Store the RNA samples in 2.0-milliliter microcentrifuge tubes at minus 80 degrees Celsius. Quantify the RNA and check the RNA for purity using a spectrophotometer.After checking the purity, analyze the RNA with a 1%agarose gel to visualize the extend of degradation. Two clear bands representing 18S and 28S rRNA should be clearly visible. If these bands are not visible, the RNA may be degraded.Next, monitor the transcript abundance in the collect aliquots of RNA compared with the spiked-in internal standard using Real-Time qPCR. Microarrays, RNA sequencing, or northern blots can also be used. Fluorescence intensities over time correspond to gene expression.While fibroblasts become contact-inhibited, the transcript-encoding Collagen 3A1, a very important gene for creating the extracellular matrix deposited during wound healing becomes more stable and does not decay as rapidly. Don't forget that working with phenol can be extremely hazardous and it is important not to let it touch your skin. After watching this video, you should have a good understanding of how to monitor transcript decay rates in proliferating and quiescent fibroblasts.Changes in transcript decay rate can be an important mechanism of gene triangulation.

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