|
| Status |
Public on Mar 31, 2007 |
| Title |
Quercetin treated cardiomyocytes for 24 hours (Q24-A). |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Rat cardiomyocytes
|
| Organism |
Rattus norvegicus |
| Characteristics |
Primary heart cells were obtained by isolation of cardiomyocytes from the ventricles of 2–4 day old Wistar rats.
|
| Biomaterial provider |
HARLAN ITALY S.R.L. Frazione Azzida 57 33049 San Pietro al Natisone UD (ITALY)
|
| Growth protocol |
Cells were seeded at a density of 1.2x106 cells/mL and were grown in Ham F10 nutrient mixture supplemented with 10 % v/v FCS and 10 % v/v HS (complete medium), 1% sodium pyruvate, and kept in this medium at 37 °C, 5 % CO2 and 95 % humidity until complete confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA), following the manufacturer’s protocol. The yield and purity of the RNA were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, Del, USA). The 28s and 18s ribosomal bands were checked using Agilent 2100 bioanalyzer with an RNA 6000 Nano LabChip Kit.
|
| Label |
Cy3
|
| Label protocol |
For each sample, mRNA was amplified starting from 1.5 μg of totRNA by Amino Allyl MessageAmp I aRNA Kit (Ambion Inc.) to obtain amino allyl antisense RNA following the method developed by Van Gelder et al. (Proc Natl Acad Sci USA 1990,87:1663-1667) Only one round of amplification was performed, according to the manufacturer’s protocol, with minor modification. Briefly: mRNA was reverse transcribed into cDNA single strand; after the second strand synthesis, cDNA was in vitro transcribed in aaRNA including an amino allyl modified nucleotide (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy3 dye (Amersham Biosciences, Piscataway, NJ - USA) able to react with the modified RNA. At least 5 μg of mRNA, for each sample, were labeled and purified with columns.
|
| |
|
| Channel 2 |
| Source name |
Rat cardiomyocytes treated with quercetin for 24 hours
|
| Organism |
Rattus norvegicus |
| Characteristics |
Primary heart cells were obtained by isolation of cardiomyocytes from the ventricles of 2–4 day old Wistar rats.
|
| Biomaterial provider |
HARLAN ITALY S.R.L. Frazione Azzida 57 33049 San Pietro al Natisone UD (ITALY)
|
| Treatment protocol |
30 μM quercetin was added to the culture medium for 24 hours.
|
| Growth protocol |
Cells were seeded at a density of 1.2x106 cells/mL and were grown in Ham F10 nutrient mixture supplemented with 10 % v/v FCS and 10 % v/v HS (complete medium), 1% sodium pyruvate, and kept in this medium at 37 °C, 5 % CO2 and 95 % humidity until complete confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Total RNA Isolation Mini Kit (Agilent Technologies, Palo Alto, CA), following the manufacturer’s protocol. The yield and purity of the RNA were measured using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., Rockland, Del, USA). The 28s and 18s ribosomal bands were checked using Agilent 2100 bioanalyzer with an RNA 6000 Nano LabChip Kit.
|
| Label |
Cy5
|
| Label protocol |
For each sample, mRNA was amplified starting from 1.5 μg of totRNA by Amino Allyl MessageAmp I aRNA Kit (Ambion Inc.) to obtain amino allyl antisense RNA following the method developed by Van Gelder et al. (Proc Natl Acad Sci USA 1990,87:1663-1667) Only one round of amplification was performed, according to the manufacturer’s protocol, with minor modification. Briefly: mRNA was reverse transcribed into cDNA single strand; after the second strand synthesis, cDNA was in vitro transcribed in aaRNA including an amino allyl modified nucleotide (aaUTP). Both dsDNA and aaRNA underwent a purification step using columns provided with the kit. Labeling was performed using NHS ester Cy5 dye (Amersham Biosciences, Piscataway, NJ - USA) able to react with the modified RNA. At least 5 μg of mRNA, for each sample, were labeled and purified with columns.
|
| |
|
| |
| Hybridization protocol |
The same quantity of differentially labeled sample and reference (0.75 μg) was put together, fragmented and hybridized to high-density rat arrays containing 22,575 (60 mer) oligo-nucleotide probes representing over 20,000 well characterized rat genes, ESTs, and EST cluster. All steps were performed using the In Situ Hybridization kit-plus (Agilent Technologies) and following the 60-mer oligo microarray processing protocol (Agilent Technologies). Then, slides were washed with the SSC wash procedure (Agilent Technologies).
|
| Scan protocol |
The array was scanned with the dual-laser microarray scanner Agilent B (Agilent Technologies) at 5 μm resolution.
|
| Description |
Data normalization was performed with the Agilent Feature Extraction software. Raw result files were then loaded into the Resolver SE System (Rosetta Biosoftware, Seattle, WA - USA) for data processing and normalization using the Agilent platform-specific error model. Replicated expression profiles were combined to form ratio experiments where each gene is associated to an expression fold-change and a p-value that assesses the statistical significance of its modulation in the treated sample compared to the control reference. Sequences with absolute fold-change >=2 and p-value <= 0.001 were considered as differentially expressed.
|
| Data processing |
Feature extraction was performed with the Agilent Feature Extraction software. LogRatio was calculated as Log of ratio channel 2/channel 1.
|
| |
|
| Submission date |
Mar 08, 2007 |
| Last update date |
Mar 09, 2007 |
| Contact name |
Cristina Angeloni |
| E-mail(s) |
[email protected]
|
| Phone |
+39 051 2091211
|
| Organization name |
University of Bologna
|
| Department |
Biochemistry
|
| Lab |
Nutrition Research Center
|
| Street address |
Via Irnerio 48
|
| City |
Bologna |
| State/province |
BO |
| ZIP/Postal code |
40126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL890 |
| Series (1) |
| GSE7222 |
Global gene expression analysis of quercetin bioactivity in cultured rat cardiomyocytes |
|
