Supplemental information and more detailed protocols are available at [46
MATα S288c (his3 leu2-3, 112 lys2 trp1 ura3-52) cells were grown in YPD+A (1% yeast extract, 2% peptone, 2% D-glucose, and 0.04 mg/ml adenine) with aeration at 30°C for 7 days (to OD600 20-24). Rapidly dividing (exponential) cells were collected after overnight growth (OD600 1-2) under the same conditions.
Cells from stationary phase and exponentially growing cultures were collected to serve as a common reference for all experiments. Several cell samples were taken prior to exposure to menadione to serve as a T0
reference. For oxidative stress menadione was added from a 500 μM stock to a final concentration of 50 μM. For the 30 minute time interval time course, cells were collected by pipette every 30 minutes for 8 hours. Cell viability was constant for at least 8 hours after exposure to this concentration of menadione (data not shown). For the second time course, an automated-sampling device [31
] was used to collect cells at 1 minute intervals for 35 minutes, with a final time point at 1 hour. For both time courses, cells were harvested and analyzed in duplicate by microarrays. For temperature upshift, cultures were grown to stationary phase at 30°C, shifted to 39°C, and cells harvested by pipette every 30 minutes for 8 hours.
A random block design [47
] was used to eliminate artificial sources of periodicity that may be introduced due to specific, constant ordering of time course samples during RNA isolation, cDNA labeling or hybridization, as well as to avoid confounding factors throughout the experiment. In a randomized block design, each time course is treated as a single block and samples within a time course were randomized for RNA isolation and re-randomized for both cDNA labeling and hybridization.
For RNA isolation (Additional data file 8), a modified Gentra protocol was used. Briefly, 20 OD600 of cells from exponential phase or 30 OD600 of cells from stationary phase cultures were lysed at 4°C in 300 μl of Cell Lysis Solution (Gentra, Minneapolis, MN, USA) using 0.5 mm glass beads (Sigma, St Louis, MO, USA) and a mechanical bead beater (Biospec Products, Bartlesville, OK, USA) at 4,800 rpm. Lysis was carried out in 6, 30-second bursts alternating with 30 seconds on ice. Samples were spun at 13,000 × g for 3 minutes at 4°C. After centrifugation, 100 μl of Protein-DNA Precipitation Solution (Gentra) was added to the supernatant and samples were incubated on ice for 5 minutes. Protein-DNA precipitate was pelleted at 13,000 × g for 3 minutes at 4°C. RNA was precipitated using 300 μl 100% isopropanol and pelleted by centrifugation. After centrifugation, RNA was resuspended in DEPC (diethylpyrocarbonate)-treated H2O and a phenol-chloroform (5:1) 'back extraction' was performed (Ambion, Austin, TX, USA). RNA was precipitated overnight in 0.1 volume of 0.5 M NH4OAc and 2.5 times the volume of 100% ethanol and subsequently purified using a Qiagen RNeasy kit (Qiagen, Alameda, CA, USA). RNA quality was evaluated using a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA).
For hot phenol extractions, 30 OD600 of cells from stationary phase cultures were resuspended in 1 ml of sodium acetate buffer (50 mM sodium acetate, 10 mM EDTA, 0.1% SDS) and an equal amount of saturated phenol (Fisher Scientific, Pittsburgh, PA, USA) at 65°C. Samples were incubated at 65°C for 10 minutes, with vortexing every 1 minute for 10 seconds. Samples were spun for 10 minutes. The aqueous phase was then transferred to 1 ml of saturated phenol, vortexed for 1 minute and spun for 10 minutes. After the previous step was repeated the aqueous phase was transferred to 1 ml of chloroform (Sigma), vortexed for 45 seconds, and spun for 5 minutes. RNA was precipitated with 0.1 volume of 3 M Na Ac pH 5.2 and 2 times the volume of 100% ethanol at -20°C for 1 hour. RNA was pelleted by centrifugation, washed with 70% ethanol, and RNA was resuspended in DEPC-treated H2O. RNA quality was evaluated using a Bioanalyzer 2100 (Agilent).
Array printing and slide treatment
UltraGAPS slides (Corning, Corning, NY, USA) were printed using an OmniGrid 100 Arrayer (GeneMachines, San Carlos, CA, USA) with SMP4 printing pins (TeleChem, Sunnyvale, CA, USA). The yeast genomic oligonucleotide set (containing 70-mers corresponding to 6,307 open reading frames; Qiagen) was resuspended in 3× SSC to a final oligonucleotide concentration of 40 μM, and used to print the slides.
During printing, the relative humidity was maintained between 50% and 52% and the ambient temperature was maintained between 21°C and 23°C. After printing, slides were UV cross-linked at 90 mJ in a UV Stratalinker 1800 (Stratagene, La Jolla, CA, USA) and baked at 80°C overnight. For validation and quality control, slides were scanned after pre-hybridization treatment (see below) to screen for spot-localized contamination [48
]; SYBR green II staining (Invitrogen, Carlsbad, CA, USA) was used to test for DNA binding; and reproducibility experiments to test slide-to-slide reproducibility were carried out prior to and in each experiment. Typically, slide-to-slide standard deviation was in the range of 0.08 log2
units for expression ratios.
Preparation of labeled cDNA
A modified direct-labeling protocol [49
] was used to fluorescently label cDNA with Cy3-dCTP or Cy5-dCTP (Amersham Biosciences, Piscataway, NJ, USA) using the Corning Microarray Technologies (CMT) Yeast Array 9/00 protocol (Corning; Additional data file 9). Total RNA (20 μg) from experimental samples were reverse transcribed to cDNA labeled with Cy5. A common reference sample RNA (20 μg of stationary phase and exponential RNAs combined at a 1:1 ratio) was reverse transcribed to cDNA labeled with Cy3 and pooled after labeling. A pooled sample of the common reference was optimized to maximize the number of genes hybridized and reduce variability throughout the array [50
] and was used for all experiments. The use of a common reference allows all experimental information to be analyzed in relationship to the same reference, allowing better normalization [51
For within-slide normalization, 10 ng of Arabidopsis thaliana CAB mRNA (Stratagene) was added to each labeling reaction to be used. Because we use a common reference and all the experimental information is in Cy5-labeled cDNA, even if the CAB mRNA labels slightly differently using Cy3 or Cy5, the normalization is consistent throughout the experiment.
Pre-hybridization and hybridization
Slides were pre-hybridized in a 250 ml glass Coplin jar for 1 to 2 hours at 42°C in a freshly prepared solution containing 50% formamide, 5× SSC, 0.1% SDS and 0.1 mg/ml bovine serum albumin fraction V (Sigma; Additional data file 10). The slides were rinsed several times with ddH2O, dipped in 100% ethanol, and dried under a 30 psi stream of N2 gas. Prior to hybridization, 22 × 30 mm Lifterslip coverslips (Erie Scientific, Portsmouth, NH, USA) were cleaned in a solution of 1 M KOH and 50% ethanol, rinsed with ddH2O and dried with 30 psi of N2 gas.
The hybridization buffer contained 50% formamide (Sigma), 5% dextran sulfate (Sigma), 5× SSC, 0.1% SDS, 0.1 mg/ml bovine serum albumin fraction V (Sigma), and 100 μg/ml salmon sperm DNA (Invitrogen). For all hybridizations, reference samples were labeled and pooled prior to hybridization. Each labeled experimental sample was combined with an aliquot of the labeled reference sample and dried down in a vacuum centrifuge.
The combined reactions for each slide were resuspended in 35 μl hybridization buffer, incubated at 95°C for 5 minutes, centrifuged for 30 seconds, and applied to the center of the coverslip that was subsequently positioned to cover the printed section of the slide. Slides were sealed in CMT hybridization chambers (Corning) and incubated at 42°C for at least 16 hours on a rocking platform. After hybridization, slides were washed as previously described [48
] and subsequently dried using a 30 psi stream of N2
Microarray scanning and data analysis
All scans were performed with 100% laser power and photomultiplier tube (PMT) settings of 630 to 700 for the 635 nm laser and 430 to 500 for the 532 nm laser using Axon 4000B (Axon Instruments, Union City, CA, USA). These settings provided the maximum signal to noise ratio while reducing the number of saturated spots on each array. Grids used to define spot circumference, location, and identity were made and aligned using GenePix Pro 6.0 (Axon Instruments). All microarray data have been submitted to Gene Expression Omnibus series accession number GSE3729.
Evaluation of correlation between biological and technical replicates
An ANOVA measurement model was used to determine variance between six independent biological and technical replicates of T0 samples (Additional data file 11). The analysis showed a standard deviation for log2 ratios of 0.08 for both biological and technical replicates, indicating that a change in gene expression greater than 1.6-fold could be viewed as statistically significant with a Type I error (or false positive rate) of ≤ 0.01.
Quantitative RT-PCR analysis
Primers were designed using Primer Express (ABI, Foster City, CA, USA) and total RNA was reverse transcribed using either oligo-dT or random hexamers as primers. The two types of primers were used to determine whether transcripts lacking a poly(A)+ tail were present in total RNA isolated from unstressed, stationary phase samples. Quantitative RT-PCR reactions contained SYBR Green PCR Master Mix (ABI), forward and reverse primers (0.6 μM each), and a cDNA template (10 ng). Reactions were done in triplicate (technical replicates) for each sample and exogenous A. thaliana
RNA (Stratagene) was used as a control (Additional data file 12). The cycle threshold (CT
) value for each reaction was determined using the ABI Prism 7000 SDS software package (ABI). CT
values were used to calculate the mean fold change of the reactions via the
method, for which 1 indicates no change in abundance [52
]. For this experiment, the mean fold change was the difference in abundance in random hexamer-primed cDNA compared with the abundance in oligo-dT-primed cDNA.
After bead beating samples were centrifuged at 13,000 × g for 3 minutes at 4°C to remove cell debris. Cell-free lysates were divided into 2 tubes and incubated on ice for 1 hour with either 17 mg/ml trypsin in 0.9% (w/v) NaCl (Invitrogen), 14 mg/ml proteinase K in 10 mM Tris pH 7.5 (Qiagen), 11 mg/ml Qiagen protease in 10 mM Tris pH 7.5 (Qiagen), or 10 mM Tris pH 7.5 alone. After incubation, the RNA isolation for both samples was completed as described above. To visually evaluate the extent of protein digestion under these conditions, proteins isolated from protease-treated and control samples were separated by electrophoresis on a 10% TBE polyacrylamide gel (Bio-Rad, Hercules, CA, USA; Additional data file 4). All experiments were done in duplicate, using biological replicates. Microarray analysis showed that RNA isolated from samples incubated in buffer for 1 hour at 4°C were essentially identical to samples processed immediately (R2 = 0.97).
RNA polymerase II mutant
a (ura3-52 rpb1-1
) RNA polymerase II temperature-sensitive mutant [35
], parental MATα
), and wild-type S288c cells were grown to stationary phase (10 days) with aeration in 100 ml of YPD+A at 24°C. Prior to exposure to menadione, all strains were incubated for 3 hours at 36°C (non-permissive conditions for rpb1-1
) prior to the collection of T0
samples. Oxidative stress was induced using menadione as described above and samples from rpb1-1
, parental and S288c cultures were harvested at 2 and 30 minutes after exposure.
Statistical analysis of transcript abundance
A normal approximation to Fisher's exact test [53
] was used to generate a p
value for testing the association between membership on two sets of gene lists.