|
| Status |
Public on Jun 01, 2010 |
| Title |
Testis_Estradiol_treatmentA2_Rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Testis_Estradiol_A2
|
| Organism |
Mus musculus |
| Characteristics |
train: CD-1
gender: Male
tissue: Testis
age: four weeks
agent: Mothers were exposed to estradiol (in ethanol) two weeks before mating, 0.04 mg/l.
|
| Biomaterial provider |
Animal Facility of “Centro de Investigaciones Biologicas” CSIC.
|
| Treatment protocol |
Mothers were exposed to estradiol for two weeks before mating, 0.04 mg/l. Then, administration was interrupted. Male offspring were sacrificed to obtain the testis four weeks after birth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from testes of animals was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s standard protocol. RNA samples were spectrophotometrically quantified using NanoDrop (NanoDrop Technologies). To minimize the effect of inter-individual expression variability we made pools of RNA from at least 3 individual RNA samples from each experimental condition (same quantity of total RNA per sample). Pools of RNA were checked for quality performing Experion analysis (Bio-Rad Laboratories).
|
| Label |
Cy5
|
| Label protocol |
Starting with total RNA from pools (Three animals), full length cDNAs were synthesized from oligo(dT) primers bearing a T7 promoter. These cDNAs were used as template for in vitro transcription by T7-RNA-Polymerase to amplify the mRNAs; following the Amino Allyl MessageAmp™ aRNA Kit (Ambion, Inc.) protocol. The amplified mRNAs (aRNA) were labeled either with Cy3 or Cy5 dyes, using Amersham CyDye Post-Labeling Reactive Dyes (Amersham Biosciences). The incorporated dye was spectrophotometrically quantified using NanoDrop (NanoDrop Technologies)
|
| |
|
| Channel 2 |
| Source name |
Testis_Control_Estradiol_A2
|
| Organism |
Mus musculus |
| Characteristics |
strain: CD-1
gender: Male
tissue: Testis
age: four weeks
agent: not exposed to chemical compounds.
|
| Biomaterial provider |
Animal Facility of “Centro de Investigaciones Biologicas” CSIC.
|
| Treatment protocol |
No treatment. Testis from four weeks mice were collected and RNA isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from testes of animals was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s standard protocol. RNA samples were spectrophotometrically quantified using NanoDrop (NanoDrop Technologies). To minimize the effect of inter-individual expression variability we made pools of RNA from at least 3 individual RNA samples from each experimental condition (same quantity of total RNA per sample). Pools of RNA were checked for quality performing Experion analysis (Bio-Rad Laboratories).
|
| Label |
Cy3
|
| Label protocol |
Starting with total RNA from pools (three animals), full length cDNAs were synthesized from oligo(dT) primers bearing a T7 promoter. These cDNAs were used as template for in vitro transcription by T7-RNA-Polymerase to amplify the mRNAs; following the Amino Allyl MessageAmp™ aRNA Kit (Ambion, Inc.) protocol. The amplified mRNAs (aRNA) were labeled either with Cy3 or Cy5 dyes, using Amersham CyDye Post-Labeling Reactive Dyes (Amersham Biosciences). The incorporated dye was spectrophotometrically quantified using NanoDrop (NanoDrop Technologies).
|
| |
|
| |
| Hybridization protocol |
Prehybridization was performed at 42ºC for 30-45 minutes in 6X SSC, 0.5% SDS and 1% BSA. Slides were rinsed five times with destilled water. Cy5 and Cy3 amplified RNA (aRNA) probes were mixed (150 pmol of each label) with 10 µg of PolyA (Sigma) and 1 µg of Cot-DNA (Invitrogen) in a final volume of 40 µl of hybridization buffer (50% formamide, 6X SSC, 0.5% SDS, 5X Denhardt’s). The probe was denatured at 95ºC for 5 minutes and applied to the slide using a coverslip. Slides were then incubated at 42ºC for 16h in hybridization chambers (Array-It). After incubation, slides were washed twice with 0.1X SSC, 0.1% SDS for 5 minutes each, three times with 0.1X SSC for 5 minutes and finally in distilled water for 10 seconds. Slides were dried by centrifugation at 563g for 1 minute.
|
| Scan protocol |
Images from Cy3 and Cy5 channels were equilibrated and captured with an Axon 4000B scanner (Axon Instruments) and spots quantified using GenePix 5.1 software (Axon). GPR files were then included in almaZen database software (Bioalma). Signals were background subtracted, and normalized by total intensity and lowess_print_tip_group.
|
| Description |
Dye-Swap replicates were performed: one slide hybridized with experimental and control targets labeled with Cy3 and Cy5, respectively; and the second slide with the same type of targets but inversely labeled. Target refers to labeled aRNA while probe refers to the oligonucleotides spotted on the microarray platform.
Imaging and data generation were carried out using a GenePix 4000B (Axon Instruments) and associated software from Axon Instruments, Inc. The microarray slides were scanned with dual lasers with wavelength frequencies to excite Cy3 and Cy5. Images were captured in TIFF files. Information extraction for a given spot was based on the median value for the signal pixels minus the median value for the background pixels to produce a gene set data file for all the DNA spots. The Cy3 and Cy5 fluorescence signal intensities were normalized.
|
| Data processing |
GPR files with raw data were introduced in almaZen database software (http://www.Bioalma.com). Signals were background subtracted, and normalized by total intensity and Print_tip_group_Lowess.
|
| |
|
| Submission date |
Feb 05, 2009 |
| Last update date |
Dec 22, 2009 |
| Contact name |
Luis A. Lopez-Fernandez |
| E-mail(s) |
llopezf.hgugm@salud.madrid.org
|
| Phone |
34 914265026
|
| Organization name |
Hospital General Universitario Gregorio Marañon
|
| Department |
Pharmacy
|
| Lab |
Pharmacogenetics and Pharmacogenomics
|
| Street address |
Dr. Esquerdo 46
|
| City |
Madrid |
| ZIP/Postal code |
28007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7509 |
| Series (1) |
| GSE14774 |
Altered gene expression profiles in testis developmental exposed to endocrine disruptors |
|
