title: LAFUGA Cattle subtr lib 18KB v1.0
status: Public on Dec 31 2005
contact.city: Lindau
contact.first: Stefan
contact.middle: Michael
contact.last: Bauersachs
contact.email: stefan.bauersachs@uzh.ch
contact.state: Zurich
contact.address: Eschikon 27 EHB 23.1
contact.department: Department for Farm Animals
contact.country: Switzerland
contact.institute: University of Zurich
organism: Bos taurus
description: Pretreatment of Animals and Collection of Endometrial Tissue Samples for the production of subtracted cDNA libraries:
  
  Ten cyclic heifers (Deutsches Fleckvieh, Simmental) between 18 and 24 months old were cycle synchronized by injecting intramuscularly a single dose of 500 µg Cloprostenol (Estrumate®; Essex Tierarznei, Munich, Germany) at dioestrus. Animals were observed for sexual behaviour (i.e., toleration, sweating, vaginal mucus) to determine standing heat, which occurred around 60 h after Estrumate injection. In addition, animals were checked with ultrasound-guided follicle monitoring. Blood samples were taken at day 20, day 0 of the estrous cycle and just before slaughtering to determine serum progesterone (P4) levels. The first group of animals was inseminated with cryo-conserved sperm (ejaculate + diluter 1:10) at estrus. Animals were slaughtered at Day 18 of gestation and intact concepti were detected in the ipsilateral uterine horns of the animals used for gene expression analysis (n=4). Control animals (n=6) were inseminated with the supernatant of centrifuged sperm and also slaughtered at day 18 of the estrous cycle to achieve an identical as possible pretreatment of both groups. No conceptus was found in control animals. Sperm was derived from the same bull. The uterus was prepared and opened longitudinally. In pregnant animals, the conceptus was localized and carefully removed. Samples were carefully cut out from the lamina propria of the intercaruncular endometrium with a scalpel and immediately transferred into an appropriate amount of RNAlater (Ambion). Tissue sample of the caudal part of the ipsilateral uterine horn were used for transcriptome analyses.Samples were stored at -20°C until further processing.
  
  Generation of Subtracted cDNA Libraries:
  The production of subtracted libraries was done according to the Suppression Subtractive Hybridization (SSH) method (Diatchenko et al. 1996). Total RNA from endometrial tissue samples of the ipsilateral caudal part of the uterus was isolated using Trizol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations. The quantity and quality of total RNA was determined by spectrometry and agarose gel electrophoresis. Sixty microgram of pooled total RNA (corresponding to approximately 1.8 µg mRNA) were used for the preparation of subtracted cDNA libraries for pregnant and control animals, respectively. The construction of the libraries was done as previously described (Bauersachs S et al. 2003; Bauersachs S et al. 2004) with the modification that no additional driver cDNA was added at the second step of subtractive hybridization. For every library 1,536 cDNA clones were randomly picked. This array (LAFUGA Cattle subtr lib 18KB v1.0) contains the cDNA clones derived from the subtracted library (18KB) enriched for cDNAs of genes upregulated in endometrium of Day 18 pregnant animals.
manufacture_protocol: 1,536 cDNA clones were randomly picked for each library and grown overnight at 37 °C in 100 µl LB medium in 96-well microtiter plates. Bacterial suspensions were diluted 20-fold in TE buffer (pH 8.0), incubated at 96 °C for 10 min, and stored at -20 °C. PCR amplification of the cDNA insertions was performed in 20 µl in 96-well cycle plates and contained 1 µl diluted bacterial cells, PCR buffer (peQLab, Erlangen, Germany), 200 pmol/µl dNTPs, 0.3 pmol/µl of the respective primers (modified T3 and T7 primers), and 1 U Taq DNA polymerase (peQLab). All PCR products were analysed by 0.8 % agarose gel electrophoresis showing that more than 95 % of the PCR reactions yielded defined PCR products. 15 µl of PCR reactions were transferred to 384-well microtiter plates (ABgene) containing 15 µl 2-fold spotting buffer (40 mmol/l Tris-HCl pH 8, 2 M NaCl, 2 mmol/l EDTA, bromophenol blue). 1,536 PCR products were spotted onto nylon membranes (Nytran Supercharge, Schleicher & Schuell, Dassel Germany) on an area of 20 x 50 mm using an Omnigrid Accent microarrayer (GeneMachines, San Carlos, California, USA) and solid pins (SSP015, diameter 0.015 inches; Telechem International, Sunnyvale, California, USA). Spotting was done 6 times for each PCR product on the same position for sufficient and equal application. 25 arrays were produced simultaneously. The cDNAs were denatured on the arrays by incubation on filter paper (Schleicher & Schuell) soaked with 0.5 N NaOH for 20 min at room temperature. DNA was fixed by baking at 80 °C for 30 min and UV cross-linking (120 mJ/cm2, XL-1500 UV Crosslinker, Spectronics Corp., New York, USA).