Additional files
Additional file 1 – Protocol for extraction of RNA
Extraction of RNA was performed on each sample using an established technique at PMCC –
‘Trizol/RNeasy total RNA extraction for human/animal tissues’.
In summary, approximately 15-30mg of tissue was crushed on dry ice, combined with 2ml of
Trizol and homogenised at room temperature using an Ultraturrax probe (John Morris
Scientific). After the addition of 400μl chloroform, the homogenate was shaken vigorously
for 30 seconds, then left to stand at room temperature for 5 minutes. Samples were then
centrifuged at 11,000rpm at 4C for 15 minutes. The clear supernatant was carefully
removed, without disturbing the interface, and transfered to a clean tube. The supernatant was
mixed by vigorous shaking with an equal volume (~1-2ml) of 70% alcohol, then transferred
to an RNeasy (Qiagen) column and placed on a vacuum. The column was washed with 700μl
Buffer RW1 (Qiagen), followed by 2 washes with 500μl Buffer RPE (Qiagen). To ensure the
membrane was clean and dry, the RNeasy (Qiagen) column was placed into a 2ml collection
tube and centrifuged in a microcentrifuge at full speed for 1 minute. The flow through and
collection tube was discarded. Finally, the column was eluted twice with 50ul RNase-free
water, with collection of the eluate in a new 1.5ml collection tube. A spectrophotometer was
used to measure RNA concentration by estimating absorbance at 260 nm and 280 nm. A ratio
of <1.8 suggested poor quality RNA and was not used. Purified RNA was precipitated in 0.1
volumes of 3 M Sodium Acetate and 2 volumes of 100% ethanol and stored in a -20ºC
freezer overnight. After centrifuging at 13,000rpm at 4C for 15 minutes, the supernatant was
discarded and the pellet washed with 500μl 70% ethanol. The pellet was then resuspended in
an appropriate volume of RNase-free water to give a final RNA concentration between 1 –
8ug/μl. A repeat assessment of RNA concentration was performed and samples were stored
in -80C until required for transcriptional studies.
Additional file 2 – Protocols for extraction of DNA
Method 1
‘Qiagen® DNeasy Mini Kit’ was used, following the method recommended for the isolation
of total DNA from animal tissues. In brief, approximately 25-50mg of frozen tumour tissue
was cut into small pieces, and placed in a 1.5ml microcentrifuge tube with 180μl Buffer ALT.
The sample was combined with 20μl proteinase K and mixed vigorously, then incubated at
55C on a rocking platform until the tissue was completely lysed (approx 3 – 4 hours). 200μl
Buffer AL was added to the lysed sample, and mixed thoroughly by vortexing (it is essential
that the sample and Buffer AL are mixed immediately to yield a homogenous solution), then
incubated at 70C for 10 minutes. After the addition of 200μl of 96–100% ethanol and
thorough mixing, the sample was transferred to a DNeasy mini spin column, placed on a
vacuum, and washed with 500 μl each of Buffer AW1 and Buffer AW2.
To ensure the DNeasy membrane was completely dry, with no residual alcohol, the mini spin
column was placed into a 2ml collection tube and centrifuged for 3 minutes at 14,000rpm.
The flow-through and collection tube was discarded and the mini spin column placed in a
new 2ml microcentrifuge tube and eluted with 200μl Buffer AE.
The elution step was repeated with a new collection tube.
DNA yield, or concentration, was determined by measuring the absorbance of the eluate at
260nm (A260), using a spectrophotometer. An A260 of 1 corresponds to a DNA
concentration of 50ug/ml. The concentration of DNA can therefore be calculated quite easily
according to the A260. The ratio of readings at 260nm and 280nm (A260/280 ratio) provides
an estimate of DNA purity with respect to contaminants that absorb UV, in particular
proteins. A ratio of 1.8 – 2.0 is desirable, indicating pure DNA. Ratios below 1.8 indicate
possible protein contamination.
Method 2 - Phenol/chloroform method
25-50mg of frozen tumour tissue was crushed or cut into small pieces, suspended in 1.2ml of
digestion buffer per 100mg of tissue, then incubated at 55C on a rocking platform until the
tissue was completely lysed (approx 12 - 18 hours). An equal volume of
phenol/chloroform/isoamyl alcohol was added to each sample and mixed well, then
centrifuged at room temperature at 13,000rpm for 10 minutes. To ensure removal of all
phenol, an equal volume of chloroform was added to the supernatant and mixed well. After
centrifuging at room temperature at 13,000rpm for 10 minutes, the supernatant was combined
with 0.5 volumes of 7.5M ammonium acetate, 2 volumes of absolute ethanol, and 1μl of
glycogen, then incubated at -20C for 1 hour. The pellet was washed with 500μl of 80%
ethanol, and then resuspended in 40μl RNase-free water or TE buffer.
Additional file 3 – Transcriptional profiling
Part 1: RNA amplification
For first strand synthesis, approximately 3µg of total RNA was combined with T7 anchored
polyT primer and Rnase free water, then incubated at 70ºC for 10 minutes, followed by the
addition of 5x Promega RT buffer, Rnase-free water, linear acrylamide, dNTP, reverse
transcriptase MMLV Rnase H-point mutant and Rnasin, and further incubation at 42ºC for 1
hour.
Second strand synthesis followed with the addition of Rnase-free water, 10x DNA
polymerase I buffer, dNTP, DNA polymerase I, E. coli DNA ligase and Rnase H, incubated
at 16ºC for 2 hours. After incubation, linear acrylamide, EDTA and phenol/chloroform were
vigorously mixed then centrifuged at 13,000 rpm for 2 minutes. The aqueous phase was
combined with 7.5M ammonium acetate and 100% ethanol, then centrifuged at 13,000 rpm at
room temperature for 20 minutes. The pellet was washed with 80% ethanol, then the resulting
double stranded DNA (dsDNA) resuspended in Rnase-free water.
Part 2: Transcription, RNA purification and concentration
In an Rnase-free PCR tube, dsDNA was combined with 10x reaction buffer, NTP mix and T7
polymerase/superase enzyme mix, then incubated at 37ºC for 4 hours. The resulting RNA
was purified using an Rneasy Miniprep Kit (Qiagen) and following the manufacturers
instructions. RNA concentration and purity was then assessed using a spectrophotometer.
Part 3: Indirect labelling of aRNA
An aliquot of 4-10μg of amplified RNA (aRNA) was transferred to a dome cap 200 µl PCR
tube (Scientific Specialties Inc.) which contained 2.5 µl of a set of exogenous RNA templates
used for internal quality control (ScorecardTM, Amersham Biosciences), then random
hexamer primer and Rnase-free water were added.
The same amount of reference RNA, taken from 11 pooled cell lines, was used for labeling to
avoid global differences in signal intensity between the test and reference (table E3.1).
Table S3.1: Components of 11-pooled cell line reference
Growth
ATCC
catalogue
Name
Description
Properties
Reference
MCF7
breast adenocarcinoma-derived cell line
adherent
ATCC HTB-22
Hs578T
breast adenocarcinoma-derived cell line (stromal-like) adherent
ATCC HTB-126
NTERA2
teratoma-derived cell line
adherent
ATCC CRL-1973
Colo205
colon tumor-derived cell line
mixed
ATCC CCL-222
adherent
ATCC HTB-161
OVCAR-3 ovarian tumor-derived cell line
UACC-62
melanoma-derived cell line
adherent
Stinson et al. (1992)
MOLT-4
T cell leukemia-derived cell line
suspension
ATCC CRL-1582
RPMI 8226 multiple myeloma-derived cell line
suspension
ATCC CCL-155
NB4+ATRA Acute promyelocytic leukemia-derived cell line
suspension
Lanotte et al. (1991)
SW872
liposarcoma-derived cell line
adherent
ATCC HTB-92
HepG2
liver tumor-derived cell line
adherent
ATCC HB-8065
No.or
After annealing at 70ºC for 10 minutes 5x RT buffer (Promega), Rnase-free water and reverse
transcriptase MMLV Rnase H-point mutant were added, and the samples incubated at 42ºC
for 2.5 hours, to facilitate reverse transcription. Each sample was then hydrolysed with the
addition of 0.25M sodium hydroxide, 0.5M EDTA and 0.2M acetic acid. Test and reference
samples were transferred to separate 1.5 ml tubes (Ependorf) containing 5 volumes (300 µl)
of PB Buffer (Qiagen PCR purification kit, Qiagen). The PCR purification protocol was then
used according to manufacturer’s instructions, with a vacuum used in place of centrifuging.
All steps were carried out at room temperature using the mini Qiaquick PCR chromatography
columns (800 µL column capacity), and separate columns were used for test and reference
samples.
Following purification, samples were not eluted and Cy-5 and Cy-3 dyes were added to test
and reference columns respectively, then incubated for 1 hour at room temperature in the
dark.
A second round of PCR purification followed, and labelled targets (Cy3-test and Cy5reference) were eluted into a single eppendorf tube which contained a blocking mix
consisting of: 3 µl tRNA (4 mg/ml), 3 µL Cot-1 DNA (10 mg/ml), 0.75 µL Poly dA (8
mg/ml) and 0.75 µl 50x Denhart’s containing herring sperm DNA that was designed to block
non-specific hybridization. The solution was then dried in a vacuum centrifuge set at 60ºC for
approximately 40 minutes.
Part 4: Hybridisation
The dried pellet was resuspended in 4.7ul of 20x SSC and 15µl deionised formamide
(filtered, 0.22 µm, Millipore), and the solution was heated at 100ºC for 3 minutes. 0.3 µl of
10% SDS (filtered, 0.22 µm, Millipore) was added, and then the denatured target was applied
to a coverslip (24mm x 40mm, Menzel-Glaser) and transferred to a microarray slide by
inverting the slide over the coverslip. The slide was immediately placed in the humidified
chamber and hybridised at 42ºC for 14-16 hours.
Part 5: Washing
Following hybridisation, slides were washed to remove excess labeled target and reagents.
All wash reagents were filtered through a 0.22 µm bottle-top filter and all steps were
performed in light-proof containers.
Coverslips were removed by immersion in 50ml of Wash 1 (0.5x SSC and 0.01% SDS) in a
glass Coplin jar for approximately 60 seconds. Slides were then placed in a darkened
container containing ~250ml of Wash 1 and agitated gently at room temperature for 1 minute.
In new darkened containers, slides were washed for 3 minutes with ~250ml of Wash 2 (0.5x
SSC), then ~250ml of Wash 3 (0.06x SSC). Wash 3 was repeated for 30 seconds prior to
removing slides and drying in a centrifuge (Hareus Megafuge) at 800rpm for 5 minutes.
Slides were scanned and then stored in a light-proof container.
Part 6: Scanning
QC metrics were followed to select slides suitable for further analysis. Scanning of
microarrays was performed using a dual laser microarray scanner (model G2505B, Agilent
Technologies). The confocal laser used excitatory wavelengths of 532 nm and 635 nm for
Cy3 and Cy5 channels, respectively.
Data was presented using Feature Extraction Software (Version 7.5, Agilent Technologies) as
a 16-bit TIFF image for each channel, and was reviewed using a pseudo-colour overlay
image of the Cy5/Cy3 channels, with red allocated to Cy5 and green to Cy3. The images
allowed for visual assessment of spot morphology, signal intensity and background or other
staining artifacts.
Genepix Pro software (version 5.1, Axon Instruments) was used to extract data from scanned
TIFF images.
Additional file 4 – Genomic profiling
Test genomic DNA was labelled with Cy3 fluorochrome, whilst reference genomic DNA
(normal human female) was labelled with Cy5 fluorochrome. Labelled test and reference
DNA were mixed with unlabelled blocking DNA, which blocks repetitive sequences in the
genome, and denatured. The mixture was then hybridised to 24K BAC array slides. Digital
images were created and computer analysed to calculate a background corrected log2 ratio of
the hybridised fluorochromes. Normalised log2 ratios provide information regarding relative
gene copy number between 2 specimens, where a log2 ratio of zero is representative of a
diploid genome.
Additional file 5 - 40 matched transcripts between GSE11117 and our
differential gene list for recurrence
UNIQID
H300020745
H200014214
H300011920
H200000387
H200005091
H300022343
H300022344
H200007681
H300021389
H200005230
H200000801
H200003990
H200007284
H200004783
H300014387
H200014044
H200017620
H300016793
H200014804
H300020043
H300006690
H200000254
H300018261
H200019855
H200006951
H300021933
H300021934
H300022641
H300022642
H300000889
H300012420
H300021781
H300005712
H300014842
H300021761
H300010014
H200002676
H200005996
H200008593
H200015687
Gene Name
KLK8
UBC
UBC
MSX1
CD81
MYL6
MYL6
PPM1E
PPM1E
GDI2
CRLS1
CLN5
PRKACG
CCBL1
CCBL1
TCEB3
HPS3
HPS3
SMARCA2
SMARCA2
SLC36A1
NCF2
NCF2
FRAP1
ST8SIA1
MDM2
MDM2
MDM2
MDM2
MX1
CENPA
CENPA
FXYD3
GPSM3
GPSM3
STIM1
NGFRAP1
APC
GHITM
PIK3CB