Female of fertile age Tissue: endometrium Cells: endothelial cells Menstrual cycle phase: proliferative
Biomaterial provider
Uppsala University Hospital
Treatment protocol
At subconfluency in passage 2, HEEC in four culture flasks per culture (biological replicate) were exposed to 50 µM o,p'-DDT for 24h. Control HEEC in four culture flasks per culture were exposed to fresh culture medium containing 0.21% DMSO. As an additional control, cells in eight culture flasks per culture were also exposed to fresh culture medium containing 0.21% DMSO. Of these eight flasks, HEEC in four culture flasks per culture (biological replicate) were used for a common control pool av control samples from all five biological replicates.
Growth protocol
HEEC were kept in 25 cm2 culture flasks (Fischer Scientific GTF, Västra Frölunda, Sweden) and grown in Microvascular Endothelial Cell Medium-2 (EGM™-2MV Bulletkit®; In vitro Sweden AB, Stockholm, Sweden) at 37ºC in an atmosphere of 5% CO2 in humidified air. Four culture flask per treatment alternative and culture (biological replicate) were used.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using RNeasy Mini Kit spin protocol (Qiagen AB, Solna, Sweden) and QiaShredder homogenizers (Qiagen AB, Solna, Sweden) according to manufacturer's instructions.
Label
Cy3
Label protocol
For each treatment and biological replicate, 500 ng isolated RNA was amplified and labelled using the Low RNA Input Linear Amplification Kit PLUS, Two-colour (Agilent Technologies Sweden AB, Kista, Sweden) and then purified using the RNeasy MinElute Cleanup Kit (Qiagen AB, Solna, Sweden). The amplification and labelling reactions were performed using the polymerase chain reaction (PCR) instrument model PTC-100 (MJ Research Inc, Waltham, MA, USA). Dye swaps were performed between the individuals to avoid dye bias between RNA from o,p’-DDT treated and untreated HEEC, and for the individual controls and the control pool. After the labelling reaction, the Nanodrop ND-1000 Spectrophotometer was used to measure the incorporation of Cy dye.
Channel 2
Source name
Primary culture HEEC, obtained in proliferative phase, individual 1-5, pooled control exposed 24 h
Female of fertile age Tissue: endometrium Cells: endothelial cells Menstrual cycle phase: proliferative
Biomaterial provider
Uppsala University Hospital
Treatment protocol
At subconfluency in passage 2, HEEC in four culture flasks per culture (biological replicate) were exposed to 50 µM o,p'-DDT for 24h. Control HEEC in four culture flasks per culture were exposed to fresh culture medium containing 0.21% DMSO. As an additional control, cells in eight culture flasks per culture were also exposed to fresh culture medium containing 0.21% DMSO. Of these eight flasks, HEEC in four culture flasks per culture (biological replicate) were used for a common control pool av control samples from all five biological replicates.
Growth protocol
HEEC were kept in 25 cm2 culture flasks (Fischer Scientific GTF, Västra Frölunda, Sweden) and grown in Microvascular Endothelial Cell Medium-2 (EGM™-2MV Bulletkit®; In vitro Sweden AB, Stockholm, Sweden) at 37ºC in an atmosphere of 5% CO2 in humidified air. Four culture flask per treatment alternative and culture (biological replicate) were used.
Extracted molecule
total RNA
Extraction protocol
Total RNA extracted using RNeasy Mini Kit spin protocol (Qiagen AB, Solna, Sweden) and QiaShredder homogenizers (Qiagen AB, Solna, Sweden) according to manufacturer's instructions.
Label
Cy5
Label protocol
For each treatment and biological replicate, 500 ng isolated RNA was amplified and labelled using the Low RNA Input Linear Amplification Kit PLUS, Two-colour (Agilent Technologies Sweden AB, Kista, Sweden) and then purified using the RNeasy MinElute Cleanup Kit (Qiagen AB, Solna, Sweden). The amplification and labelling reactions were performed using the polymerase chain reaction (PCR) instrument model PTC-100 (MJ Research Inc, Waltham, MA, USA). Dye swaps were performed between the individuals to avoid dye bias between RNA from o,p’-DDT treated and untreated HEEC, and for the individual controls and the control pool. After the labelling reaction, the Nanodrop ND-1000 Spectrophotometer was used to measure the incorporation of Cy dye.
Hybridization protocol
Pre-hybridization buffer (5 x sodium chloride sodium citrate [SSC, VWR International AB, Stockholm, Sweden], 5 x Denhardt’s solution [Sigma-Aldrich Sweden AB, Stockholm, Sweden], 0.02 µg/µl tRNA [Invitrogen AB, Lidingö, Sweden], 0.5% sodiumdodecylsulfate [SDS, VWR International AB, Stockholm, Sweden] and 50% formamide [Sigma-Aldrich Sweden AB, Stockholm, Sweden]) was applied to each array under a hybrislip (22 x 60 mm, Sigma-Aldrich Sweden AB, Stockholm, Sweden). Incubation was performed in an Hybridization Cassette ArrayIt™ (TeleChem International Inc ArrayIt, Sunnyvale, CA, USA) at 42°C for 45 min at 100% humidity in a Heating Circulator model 8201 (PolyScience, Niles, Il, USA). Each array was then transferred to a 50 ml centrifugation tube containing double distilled water. The arrays were washed five times by repeatedly turning the tubes upside down for 30 seconds each time. Hybridization and post-hybridization washes of the slides were performed using the Pronto! Hybridization Kit (Corning Life Sciences, Noricon AB, Helsingborg, Sweden). The Hybridization Cassette ArrayIt™ and Erie Scientific Lifterslip (25 x 60 mm, Histolab, Västra Frölunda, Sweden) were used for hybridization of the slides at 42°C for 18-20 hours. After hybridization, the slides were washed in the three wash buffers described in the Pronto! Hybridization Kit.
Scan protocol
The slides were scanned in a ScanArray Express HT microarray scanner ([Packard BioScience] Perkin Elmer Life Sciences Inc, Boston, MA, USA). The scanning was performed at two different voltage values for the photo multiplier tube to get a wide dynamic range of signals (high or low). The laser value default was 99%. The tiff files from the scanned arrays were gridded using the software Spotreader v 1.3.1 (Niles Scientific, Portola Valley, CA, USA).
Description
Biological replicate 3 of 5. Control HEEC vs pooled control HEEC from replicate 1-5, harvested after passage 2.
Data processing
The BioArray Software Environment BASE (Saal et al., 2002) and the Linnaeus Center of Bioinformatics Data Warehouse (LCB-DWH) (Ameur et al., 2006) were used for microarray data storage and analysis. Data was normalized using local background subtraction and within arrays using the print-tip lowess method (Yang et al., 2002).
