Experimental design
These experiments were designed to study the effect of heat shock and cold shock on
expression profiles of a hyperthermophilic archaeon after a twenty-minute temperature
shock. When cells entered the middle of the exponential growth phase at the optimal
growth temperature of 85C, they were transferred from an 85C water bath to a 95C
water bath or a 65C water bath.
Two biological replicates were obtained under both heat shock and cold shock
conditions. For each biological replicate, we performed a total of six technical replicates
consisting of three pairs of flip-dye experiments. As for the reference sample, a pooled
population of cDNA from three biological replicates consisting of cells growing in the
mid-exponential phase at 85C was used. All hybridizations were performed against this
reference.
Samples used, extract preparation and labeling
Methanococcus jannaschii was obtained from the Oregon Collection of Methanogens
(David Boone). Cells were cultivated in 125ml clear serum bottles (Wheaton) containing
30 ml of media as described previously (Miller et al., Appl Environ Microbiol 1988) with
Na2S as the reducing agent. To inoculate the media, 3ml of culture stored anaerobically at
room temperature was added, and the bottle was pressurized to 30 psi with a 4:1 v/v
mixture of H2 and CO2 substrate.
Bottles were placed in a reciprocal shaking water bath (Precision Scientific Model 25)
at 200 oscillations per min and 85C. Growth was followed by measurement of optical
density at 660 nm (Tsao et al., Biotechnol Bioeng 1994). In the mid-exponential phase,
serum bottles were transferred from the 85C water bath to another water bath agitating at
200 oscillations per min and 65C or 95C. After twenty minutes, 20ml of cells were
harvested and RNA extracted.
Cells were passed through a 0.45 m nitrocellulose filter (Millipore) for collection.
The filter was placed into cold phenol with 1% SDS and 0.1M Na-acetate and bead-beat
(BioSpec Products, Inc.) for 5 minutes at full speed. The aqueous layer was removed and
re-extracted with phenol, and the sample was then precipitated overnight at -20C with
3M sodium acetate and isopropanol. Precipitated RNA was re-suspended and DNasetreated (Invitrogen). The RNA was purified with the RNeasy kit (Qiagen), and RNA
quality was assessed using the BioAnalyzer 2100 (Agilent).
RNA was amino-allyl labeled with NHS-Cy dyes. 2 g of RNA and 2 l of random
hexamers (Invitrogen) were combined, incubated at 70C for 10 minutes, and snap
freezed in a dry-ice/ethanol bath for 30 sec. cDNA synthesis was carried out at 42C
overnight with SuperScript II (Invitrogen), 0.01M DTT, and 0.012 aa-dNTP mix (12.5
mM dATP/dCTP/dGTP (Invitrogen), 4.16 mM dTTP (Invitrogen), and 8.33 mM aadUTP (Ambion)). RNA template was hydrolyzed with 10 l 1M NaOH and 10 l 0.5M
EDTA followed by incubation at 65C for 15 min. cDNA was separated from reaction
reagents using a Microcon 30 spin column (Millipore) and lyophilized to dryness using a
Speed Vac (Savant). For NHS-Cy dye coupling, samples were re-suspended in 4.5 l
0.1M sodium carbonate pH 9.0 and 4.5 l of either NHS-Cy3 or NHS-Cy5 (Amersham
Pharmacia) and allowed to couple to the cDNA for one hour at room temperature in the
dark. The coupling reaction was quenched with 4.5 l hydroxylamine and incubated in
the dark for 15 minutes at room temperature. 35 l of 100 mM sodium acetate pH 5.2
was added ,and unincorporated dye was removed using the QIAquick PCR purification
kit (Qiagen). Coupled targets were analyzed using a spectrophotometer (Beckman) to
determine how much dye is incorporated and how many nucleotides there is per
incorporated dye. Targets with dye incorporation over 150 pmol and nucleotides per dye
ratios below 50 were used for subsequent hybridization. These Cy3 and Cy5-labeled
cDNA were lyophilized to dryness in a Speed Vac (Savant).
Hybridization procedures and parameters
Slides were pre-hybridized in filter-sterilized buffer (5 SSC, 0.1% SDS, 10 mg/ml
BSA) at 42C for at least one hour prior to use. Slides were then washed in MilliQ H2O
for ten minutes, in isopropanol for six minutes, and spun dry. Lyophilized, labeled cDNA
targets were re-suspended in 30 l of hybridization buffer (50% formamide, 5 SSC,
0.1% SDS, 0.2 mg/ml BSA) for 15 min at room temperature, and Cy5 and Cy3-labeled
targets were combined. Targets were then heated for 10 min at 95C and cooled on ice
for 30 sec. A clean 25mm  60 mm glass lifterslip (Erie Scientific) was placed onto each
pre-hybridized slide, and target was applied using capillary action. Slides were placed in
hybridization chambers (Corning) and allowed to hybridize at 42C overnight.
Post-hybridization washes were performed in an aluminum-foil wrapped glass dish
(VWR) on an orbital shaker. The first wash was performed in 1 SSC, 0.1% SDS at 42C
for 5 min. The second wash was performed in 0.1 SSC, 0.1% SDS for 5 min and the
final three washes were performed in 0.1 SSC for 5 min at room temperature.
Measurement data and specifications
Slides were scanned on a GenePix 4000 dual-color confocal laser scanner (Axon)
with PMT values set between 700 and 850. Paired 16 bit tiff files at 532 nm and 635 nm
were saved for each slide. Spots were identified using SpotFinder (v2.2.2, TIGR) with an
output of local background-subtracted Cy3 and Cy5-values. Data for spots with more
than fifty non-saturated pixels, a spot area/spot perimeter ratio of greater than r/2
(r=radius of spot), and 50% of pixel spot intensities greater than 2*median(background)
were considered good and kept for analysis. Another criterion for data usability was PCR
scores. PCR products were run on gels and scored into several categories: failed=0,
strong=1, weak=4, smear=5 or misprime=6 (see “Mj cDNA array description file.xls” for
this information). Spots containing PCR products scored as strong or weak and passing
the SpotFinder criteria were normalized.
Normalization and transformation was performed using MIDAS (v2.16, TIGR). Data
was print-tip lowess normalized with a smoothing parameter of 0.33 (the percent of data
used to calculate the lowess factor) and then standard deviation regularized across printtip groups (so that each group has the same spread). Pairs of flip-dye data (here called file
1 and file 2 as an example) were then subjected to a flip-dye consistency check in which
log2((R1*R2)/(G1*G2)) values are calculated (where R1/R2=Cy5-intensity values in file
1 and 2, respectively, and G1/G2=Cy3-intensity values in file 1 and 2, respectively) and
data outside of two standard deviations are zeroed out. Finally, hybridization data within
either the heat shock or cold shock experiments were standard deviation regularized
across slides. The output files contain standard deviation regularized Cy3 and Cy5-values
that are the geometric means of values found in each pair of flip-dye files (Cy3=G1*R2
and Cy5=R1*G2).
Within both the heat and cold shock experiments, we are now left with six files (two
biological replicates with three pairs of dye-swap experiments that have been combined
in one data file). These data were inputted into MEV (v2.2, TIGR) for significance
analysis. T-values were calculated for spots with data in at least four of the six data files.
Expression ratios meeting the unadjusted p-value cutoff for the normal t-distribution of
0.01 were outputted. For data in the paper, in-slide replicate data for genes meeting the pvalue cutoff of 0.01 were arithmetically averaged together to report a fold change for
each gene. Genes with fold-changes of greater than or equal to two were considered
differentially-expressed. The smallest and largest p-value out of all the in-slide replicate
data is also reported for each gene. Data meeting these cutoffs is contained in the file
“Genes_meeting_2-fold_and_p_less_than_0.01_cutoff.xls.” See Table 1 for the list of
other outputted data files.
Array Design
cDNA spotted glass arrays were printed at the Institute for Genomic Research (TIGR)
as follows. Primers (Invitrogen) were designed for all 1,738 ORFs. PCR reactions were
performed with 1 ng of genomic DNA and Platinum Taq polymerase (Invitrogen).
Products were detected on agarose gel and cleaned and concentrated using 96-well PCR
filter plates (Millipore). Resuspended products were again detected on agarose gel and
scored into several categories: failed, strong, lost-on-filter, weak, smear, or misprime.
Products scored as “lost-on-filter” were reamplified and precipitated using EtOH and 3M
sodium acetate at -80C. Only data for spots scored as strong or weak were used in the
array analysis (about 99% of the ORFs).
PCR products were arrayed into 384-well plates with 50% DMSO (Sigma). Ultra
Gaps slides (Corning) were then printed with these products using a MD-3 robot
(Amersham Pharmacia) with print-tips arrayed in a 2 by 12 format. Eight copies of genes
participating in methanogenesis and four copies of the rest of the genome were printed.
Printed slides were placed in metal slide racks (VWR) and baked at 80C for two hours.
Table 1. Name of files outputted in each step of the array analysis. 85E = 85C midexponential sample. 65-1 and 65-2 = two biological replicates for the 65C cold shock
(65TS). 95-1 and 95-2 = two biological replicates for the 95C heat shock (95TS). There
are six technical replicates for each biological replicate consisting of three sets of flip-dye
pairs. Tav files are arranged as follows: 1st six columns are the slide coordinates, 7th and
8th columns are the Cy3 and Cy5 values, respectively, 9th and 10th columns are the
Spotfinder flags (see Spotfinder documentation, TIGR), 11th column is the ORF, 12th
column is the PCR score, and the 13th column is the common name. In the Spotfinder
outputs, 1st sample in the file name corresponds to the Cy3 value and 2nd sample in the
file name corresponds to the Cy5 value. In the MIDAS outputs, the 7th column consists of
the temperature shock sample and the 8th column consists of the 85C exponential sample.
Spotfinder
MIDAS
MEV
MJ_7884(85E_vs_65-1).tav 1st_set_MJ_7884(85E_vs_651)_MJ_7881(65-1_vs_85E).tav
MJ_7881(65-1_vs_85E).tav
MJ_3987(85E_vs_65-1).tav 2nd_set_MJ_3987(85E_vs_651)_MJ_3985(65-1_vs_85E).tav
MJ_3985(65-1_vs_85E).tav
MJ_7939(85E_vs_65-1).tav 3rd_set_MJ_7939(85E_vs_651)_MJ_7938(65-1_vs_85E).tav
MJ_7938(65-1_vs_85E).tav
65C_significant_genes.xls
MJ_7885(85E_vs_65-2).tav 1st_set_MJ_7885(85E_vs_652)_MJ_3611(65-2_vs_85E).tav
MJ_3611(65-2_vs_85E).tav
MJ_3941(85E_vs_65-2).tav 2nd_set_MJ_3941(85E_vs_652)_MJ_3942(65-2_vs_85E).tav
MJ_3942(65-2_vs_85E).tav
MJ_7941(85E_vs_65-2).tav 3rd_set_MJ_7941(85E_vs_652)_MJ_7940(65-2_vs_85E).tav
MJ_7940(65-2_vs_85E).tav
MJ_3608(85E_vs_95-1).tav 1st_set_MJ_3608(85E_vs_951)_MJ_3609(95-1_vs_85E).tav
MJ_3609(95-1_vs_85E).tav
MJ_3986(85E_vs_95-1).tav 2nd_set_MJ_3986(85E_vs_951)_MJ_3984(95-1_vs_85E).tav
MJ_3984(95-1_vs_85E).tav
MJ_7777(85E_vs_95-1).tav 3rd_set_MJ_7777(85E_vs_951)_MJ_7776(95-1_vs_85E).tav
MJ_7776(95-1_vs_85E).tav
95C_significant_genes.xls
MJ_3612(85E_vs_95-2).tav 1st_set_MJ_3612(85E_vs_952)_MJ_3610(95-2_vs_85E).tav
MJ_3610(95-2_vs_85E).tav
MJ_3983(85E_vs_95-2).tav 2nd_set_MJ_3983(85E_vs_952)_MJ_4008(95-2_vs_85E).tav
MJ_4008(95-2_vs_85E).tav
MJ_7978(85E_vs_95-2).tav 3rd_set_MJ_7978(85E_vs_952)_MJ_7937(95-2_vs_85E).tav
MJ_7937(95-2_vs_85E).tav