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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

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

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.

1 2 3 4 5

Summary of Contents

Page 1 - Supplemental Methods

1 SUPPLEMENTAL MATERIAL Supplemental Methods Human samples DNA methylation patterns from human femoral artery atherectomy samples (n=22) were compare

Page 2

2 Library preparation for sequencing was a modification of the “multiplexed paired end ChIP protocol’’ (Illumina, San Diego, California, USA). Sample

Page 3

3 sample. The three normalizing genes were selected from a total of six housekeeping genes (ACTB, B2M, GUSB, PPIA, RPLPO, TUBB). The primers (table b

Page 4

4 Secondary antibodies for immunohistochemistry were from Vector Laboratories (horse anti-mouse HRP-conjugated, 1 : 200, Cat# BA-2000 and horse anti-

Page 5 - SUPPLEMENTAL REFERENCES

5 SUPPLEMENTAL REFERENCES 1. Sixt S, Rastan A, Beschorner U, et al. Acute and long-term outcome of silverhawk assisted atherectomy for femoro-poplite

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