|
| Status |
Public on Jan 16, 2009 |
| Title |
LAD coronary atherosclerotic plaque gene expression Low scanning Patient 8 (Technical replicate B) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Left anterior descendent (LAD) coronary, plaque >75% stenosis
|
| Organism |
Homo sapiens |
| Characteristics |
patient number: 8
sex: M
age (years): 68
tissue: LAD
pathology: Ischemic
comorbidities: -
pharm. therapies: Diuretics, Anti-arrhythmics, Hypertension regulators
plasma lipid/stenosis: +/ >75%
|
| Biomaterial provider |
Cardio-Chirurgic Section of the University Hospital Borgo Trento (Verona, Italy).
|
| Treatment protocol |
18 patients were recruited at the Cardio-Chirurgic Section of the University Hospital Borgo Trento (Verona, Italy) for heart transplantation or by-pass surgery. According to clinical diagnosis and angiography, patients were subdivided in atherosclerotic with at least 75% stenosis and non-atherosclerotic controls. Before and during heart transplantation, cases were subjected to standard oral medication with aspirin.
LAD coronaries were dissected from diseased hearts immediately after surgical removal, cleaned from fatty and cardiac muscle tissues and starved in RNAlater Solution (Ambion) for further microscopy and gene expression analysis. 5-μm-thick serial sections of paraffin included coronaries fragments were used for hematoxylin-eosin staining and microscopic analysis.
Visible plaques and LAD controls, adjacent to the segment utilized for histochemical analysis, were dissected from the entire coronary fragment and used separately for total RNA extraction with TRIzol (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol method (Invitrogen). The procedure was:
1. Homogenization of the coronary fragments, dimensionally homogeneous, by the IKA® Werke (GmbH & Co.) homogenizer;
2. Incubation, at room temperature, for 15 minutes;
3. Addition of 0.2ml/1ml Trizol and shake for 1 minute;
4. Leave in ice for 15 minutes and then centrifuge at 4º C for 20 minutes at 14,000 X g;
5 Transfer the supernatant to 1.5 ml microfuge non-stick tubes and add an equal volume of isopropanol to precipitate the RNA;
6. Incubate at -20º C for one hour and centrifuge at 4º C for 20 minutes at 14,000 X g;
7. Discard the supernatant and wash with ethanol 75% three times centrifuging every time for 15 minutes at 4º C at 14,000 X g;
8. Pellet was resuspended in ultraPURE™ distilled water DNase, RNase Free (Gibco).
|
| Label |
Cy5
|
| Label protocol |
Extracted total RNA was quantized by UV adsorption in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and analyzed for quality by capillary electrophoresis in an Agilent 2100 Bioanalyzer. All RNAs utilized in this study presented RIN at least 7.
1ug of total RNA for each studied patient and for the control pool obtained by mixing equal aliquots of each control coronaries was linearly amplified with with the MessageAmp™ aRNA amplification kit (Ambion). 5ug of the amplified RNA (aRNA) have been dried for 12 hours using a Heto Lyolab 3000 freeze-dryer (Heto-Holten A/S) and coupled with Cy3 and Cy5 post-labeling dye (Amersham, GE Healthcare). Coupling was performed for 45 minutes at room temperature. Unincorporated dyes were eliminated using MEGAclear™ Kit (Ambion). Labeled aRNA was eluted in ultraPURE™ distilled water DNase, RNase Free (Gibco) and quantized in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| Channel 2 |
| Source name |
Left anterior descendent (LAD) coronary, without plaque (Controls pool). Patients presented Dilated Cardiomyopathy.
|
| Organism |
Homo sapiens |
| Characteristics |
Pool derived from 10 LAD control coronaries without plaque. For description of patients pool composing see patients supplementary data table.
|
| Biomaterial provider |
Cardio-Chirurgic Section of the University Hospital Borgo Trento (Verona, Italy).
|
| Treatment protocol |
18 patients were recruited at the Cardio-Chirurgic Section of the University Hospital Borgo Trento (Verona, Italy) for heart transplantation or by-pass surgery. According to clinical diagnosis and angiography, patients were subdivided in atherosclerotic with at least 75% stenosis and non-atherosclerotic controls. Before and during heart transplantation, cases were subjected to standard oral medication with aspirin.
LAD coronaries were dissected from diseased hearts immediately after surgical removal, cleaned from fatty and cardiac muscle tissues and starved in RNAlater Solution (Ambion) for further microscopy and gene expression analysis. 5-μm-thick serial sections of paraffin included coronaries fragments were used for hematoxylin-eosin staining and microscopic analysis.
Visible plaques and LAD controls, adjacent to the segment utilized for histochemical analysis, were dissected from the entire coronary fragment and used separately for total RNA extraction with TRIzol (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with TRIzol method (Invitrogen). The procedure was:
1. Homogenization of the coronary fragments, dimensionally homogeneous, by the IKA® Werke (GmbH & Co.) homogenizer;
2. Incubation, at room temperature, for 15 minutes;
3. Addition of 0.2ml/1ml Trizol and shake for 1 minute;
4. Leave in ice for 15 minutes and then centrifuge at 4º C for 20 minutes at 14,000 X g;
5 Transfer the supernatant to 1.5 ml microfuge non-stick tubes and add an equal volume of isopropanol to precipitate the RNA;
6. Incubate at -20º C for one hour and centrifuge at 4º C for 20 minutes at 14,000 X g;
7. Discard the supernatant and wash with ethanol 75% three times centrifuging every time for 15 minutes at 4º C at 14,000 X g;
8. Pellet was resuspended in ultraPURE™ distilled water DNase, RNase Free (Gibco).
|
| Label |
Cy3
|
| Label protocol |
Extracted total RNA was quantized by UV adsorption in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and analyzed for quality by capillary electrophoresis in an Agilent 2100 Bioanalyzer. All RNAs utilized in this study presented RIN at least 7.
1ug of total RNA for each studied patient and for the control pool obtained by mixing equal aliquots of each control coronaries was linearly amplified with with the MessageAmp™ aRNA amplification kit (Ambion). 5ug of the amplified RNA (aRNA) have been dried for 12 hours using a Heto Lyolab 3000 freeze-dryer (Heto-Holten A/S) and coupled with Cy3 and Cy5 post-labeling dye (Amersham, GE Healthcare). Coupling was performed for 45 minutes at room temperature. Unincorporated dyes were eliminated using MEGAclear™ Kit (Ambion). Labeled aRNA was eluted in ultraPURE™ distilled water DNase, RNase Free (Gibco) and quantized in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).
|
| |
|
| |
| Hybridization protocol |
Ranges along 350 and 300 pico-moles and 2.7 and 3.0 µg of aRNA labeled with Cy3 and Cy5 were co-precipitated with the ammonium acetate and ethanol. Pellet was resuspended in 110 µl with the hybridization solution composed by SSC 5X, SDS 0.1%, Formamide 25% and salmon sperm purified DNA 100 ng/µl and carried out in an automatic hybridization station (ArrayBooster, Advalytix) at 48° C for 30 hours in the competitive hybridization. Microarray slides, before hybridization, have been blocked with the pre-hybridization solution (SSC 5X, SDS 0.1%, salmon sperm purified DNA 100 ng/ul and Denhardt's solution 5X) for 8 hours at 48° C. After the hybridization slides were washed at room temperature with 1X SSC 0.2% SDS for 4 minutes one time; 0.1X SSC 0.2% SDS for 4 minutes one time; 0.2X SSC for 3 minutes two times each with new solution and 0.1X SSC for 3 minutes one time.
Slides were dried centrifuging 1 minute at 800 X g at 20° C.
|
| Scan protocol |
After stringent washings, fluorescence left in the microarray slides were read with the ScanArray LITE confocal laser scanner (PerkinElmer) with 5 µm resolution. Each slide was subjected to three consecutive scans at low, medium and high settings of laser and photomultiplier. This protocol allows for a wide dynamic range of detection spectrum of fluorescent spots in the microarray like proposed by Skibbe et al. (Bioinformatics. 2006;22(15):1863-1870) . Low, intermediate and high intensity scans images were quantified with ScanArray Express (PerkinElmer) software using the fixed circle method.
|
| Description |
Scanning parameters are often overlooked when optimizing microarray experiments. A scanning approach that extends the dynamic data range by acquiring multiple scans of different intensities has been utilized in these experiments (see data processing).
|
| Data processing |
Data from each of three scan intensities (low, medium, high) were analyzed separately. Fluorescence intensity was determined with the ScanArray Express software (PerkinElmer) using fixed circle method and median spot intensity background subtracted was normalized in the MIDAW tool using sequentially global mean normalization and local mean normalization (LOWESS) methods. Lowess normalization was performed without distinguishing between different sub-arrays.
Normalized log2(patient_ch1/controls_ch2) values were filtered according to the median of the value calculated in the empty array position for the average of the Ch1% > BKG + 1 SD and Ch2% > BKG + 1 SD values. Ch1% or Ch2% > BKG + 1 SD value is the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity. Processing for data filtering is here summarized:
1. calculate average for Ch1% > BKG + 1 SD and Ch2% > BKG + 1 SD for each gene;
2. calculate median of the average calculated in point 1 for the empty position (see table description for experiments filtering on supplementary data table);
3. exclude normalized gene expression values presenting average calculated in point 1 under median calculated in point 2 for each experiment;
4. generated expression matrix was loaded in to TIGR MeV software and filtered for missing values along experiments. Only genes presenting calculated value in all experiment but one were used to identify differentially expressed genes by SAM software;
5. differentially expressed genes calculated at the point 4 for each scan type (high, medium and low) were integrated.
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| |
|
| Submission date |
Apr 08, 2008 |
| Last update date |
Apr 23, 2010 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
stefano.cagnin@unipd.it
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6647 |
| Series (1) |
| GSE11138 |
LAD coronary atherosclerotic plaque gene expression |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized (global mean and local mean normalization (LOWESS) methods) and filtered data. Data were expressed as logarithm base 2 of Ch1/Ch2. |
| Exp.22-VALUE-NOT-FILTERED |
Normalized (global mean and local mean normalization (LOWESS) methods) not filtered data. Data were expressed as logarithm base 2 of Ch1/Ch2. |
| Ch1 Median - BKG |
Raw data. Median intensity channel 1 background subtracted. |
| Ch2 Median - BKG |
Raw data. Median intensity channel 2 background subtracted. |
| Ch1 % > BKG + 1 SD |
Raw data. Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity in the channel 1. |
| Ch2 % > BKG+ 1 SD |
Raw data. Percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity in the channel 2. |
| AVERAGE Ch1%>BKG+1SD and Ch2%>BKG+1SD |
Calculated average for the values Ch1%> BKG + 1SD and Ch2%> BKG + 1SD. |
