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

Summary

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

Abstract

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.

Introduction

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 processes^1,2,3,4. 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 huma....

Protocol

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 different cultures of cells. In our experience, one 10 cm (diameter) tissue culture dish (one plate) provides sufficient RNA for analysis. If needed, m....

Results

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 .......

Discussion

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

Materials

List of materials used in this article

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