title: Abeta-activated Microglia Part 1
status: Public on Oct 29 2003
contact.city: South San Francisco
contact.first: Scott
contact.middle: Curtis
contact.last: Lohr
contact.email: slohr@agyinc.com
contact.state: CA
contact.address: 290 Utah Ave.
contact.department: Bioinformatics
contact.country: USA
contact.web_link: http://www.agyinc.com
contact.institute: AGY Therapeutics Inc.
contact.phone: 650-228-1140
organism: Homo sapiens
description: RNA Preparation
  Total RNA from abeta stimulated cells was isolated using Tri Star reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).  The resulting RNA was treated with RNAse free DNAse (Ambion, Austin, Texas) for 40 minutes at 37.C.  Isolation of mRNA was carried out using Oligotex (Qiagen, Chatsworth, CA).
  Library Generation
  To construct the subtractive and normalized cDNA libraries, reverse transcription and second strand synthesis were performed using the Superscript II reverse transcription kit (Invitrogen, Carlsbad, CA) with an anchored Oligo dT (30) primer. Subtractive and normalized cDNA libraries were generated using suppression PCR.
  Array Creation
  Subtracted and normalized cDNA libraries were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), and plated. Single colonies were picked into 384 well plates using an automated colony picker (Q-Pix, Genetix, Cambridge, UK) to grow overnight and subsequently serve as templates for PCR. PCR products were spotted on Nylon membranes using a 384 pin head arrayer (Q-bot, Genetix, Cambridge, UK). The spotted PCR product on all filters were cross-linked to the membrane by UV light. Filters were neutralized and denatured using standard protocols.
  Library Description
  SL00097:3h abeta stimulated
  SL00098:6h abeta stimulated
  SL00099:12h abeta stimulated
  Related Platforms
  GPL562