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SUPPLEMENTAL MATERIAL
Supplemental Methods
Human samples
DNA methylation patterns from human femoral artery atherectomy samples (n=22) were compared
to normal mammary artery samples (n=9). Atherectomy samples were collected at vascular
atherectomy operations19. The nine normal samples represent trimmed ends of mammary arteries
isolated during cardiac bypass surgery. Average age (±SD) of the patients for plaques and normal
arteries were 70,9 ±7,3 and 67,2 ± 9,1 years, respectively (Table1). No significant differences were
found in major cardiovascular risk factors between atherosclerotic and control samples. The studies
were approved by Local Ethical Committee of Kuopio University Hospital under identification
number 53/2011 and the subjects gave informed consent.
Sample preparation
One half of the atherectomy samples was snap frozen in liquid nitrogen and the second half was
processed for paraffin embedding. Total RNA, DNA and protein were isolated from frozen samples
by the Trizol method (Cat# 15596-018, Life technologies, Paisley, United Kingdom).Total RNA used
for microarray analysis was repurified using RNeasy silica membrane columns (Cat# 74106; Qiagen
AB, Sollentuna, Sweden). Total RNA integrity was checked on the Agilent 2100 (Agilent, Santa Clara,
California, USA), and only samples with RNA integrity number over 5 were used for gene expression
analysis.
Dissolved DNA samples were repurified using DNeasy columns (Cat# 69506, Qiagen AB, Sollentuna,
Sweden). DNA concentration was measured on a Qubit 2.0 Fluorometer (Cat# Q32866, Invitrogen,
Carlsbad, California, USA) with PicoGreen assay kit(Cat# P7589, Invitrogen) on 480/520 nm. RNA
concentration was estimated on Nanodrop 2000 UV-VIS spectrophotometer (Thermo Scientific,
Wilmington, Delaware, USA) spectrophotometer at 260 nm wavelength.
Protein samples from Trizol procedure were further purified as described
2
and after solubilzation in
4M urea, the protein concentration was estimated by bicinchoninic acid (BCA) protein assay (Cat#
23227, Thermo Scientific, Wilmington, Delaware, USA).
DNA methylation analysis
DNA samples were fragmented on Covaris S2 with following settings: duty cycle 10%, intensity 5, 200
cycles per burst during 190 sec to obtain fragments with an average length of 200 bp. The power
mode was frequency sweeping, temperature 6 – 8
o
C, water level 12. Maximum 3 μg was loaded in
130 μl TE in a microtube with AFA intensifier (Covaris, Woburn, Massachusetts, USA). Fragment
distribution was checked on a high sensitivity chip on the Agilent 2100 (Agilent). Methylated DNA
fragments were captured using the MethylCap kit (Diagenode AF-100-0048, Belgium). The
concentrations of the fragmented and captured DNA were determined on a Fluostar Optima plate
reader (BMG Labtech, Offenburg, Germany) with the PicoGreen dsDNA assay kit (P7589, Invitrogen)
on 480/520 nm.
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