title: Porcine testis cDNA microarray 060717
status: Public on Jun 02 2007
contact.city: Chunan, Miaoli
contact.first: Wen-chuan
contact.last: Lee
contact.email: wen-chuan@mail.atit.org.tw
contact.address: 52 Ke-dung 2nd Rd.
contact.department: Division of Biotechnology
contact.country: Taiwan
contact.institute: Animal Technology Institute Taiwan
organism: Sus scrofa
description: GAPS II, Corning
manufacture_protocol: 1. Two porcine testes, from mature pigs under heat stress or not, were collected for cDNA libraries construction with SuperScript Cloning System according to the manufacturer’s instructions (Invitrogen).
  2. A total of 27 072 cDNA clones were randomly picked from two cDNA librabies for EST sequencing.
  3. Plasmids from 12 748 nonredundant cDNA clones were transferred to 96-well microplates.
  4. The cDNA were amplified by PCR using T7 primer (5’-TAATA CGACT CACTA TAGGG-3’) and SP6 primer (5’-CATAC GATTT AGGTG ACACT ATAG-3’) in a final volume of 100 μL.
  5. Transfer 3 μL of PCR products for gel electrophoresis analysis.
  6. PCR products with yield higher than 20 ng/μL were rearranged to new 96-well microplates.
  7. PCR products were purified with Montage PCR96 plate (Millipore) under vacuum and resuspended in 100 microliter of Milli-Q water.
  8. The amplicons were evaporated under vacuum at mind temperature and resuspended in 20 μL of 50% DMSO.
  9. Transfer the probes into 384-well microplates.
  10. A total of 9 944 unique Expressed Sequence Tags (ESTs) were selected for cDNA microarray construction.  Twenty control spots (Universal ScoreCard, GE Healthcare, USA) were included in the probes set.
  11. All the probes were spotted onto GAPS-II glass slides (Corning) using the OmniGrid Accent microarrayer (Genomic Solutions).
  12. The spotted cDNA microarray was cross-linked with UV at 1200 mJ/cm2 and stored in a desiccator.
  13. The quality of spotted cDNA microarray was assessed by PARAGON DNA Microarray QC Stain Kit (Invitrogen)