> Genes Defined by SAGE
SAGE Library Description
LIBRARY NAME: 1. SAGE_CPDR_LNCaP-C;
2. SAGE_CPDR_LNCaP-T
LIBRARY DESCRIPTION: LNCaP cells (Item Number CRL-1740,
American Type Culture Collection, Rockville, MD) were used for generating
SAGE libraries. LNCaP cells were maintained in RPMI 1640 (Life Technologies,
Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS,
Life Technologies, Inc., Gaithersburg, MD) and experiments were performed
on cells between passages 20 and 30. For the studies of androgen regulation,
charcoal/dextran stripped androgen-free FBS (cFBS, Gemini Bio-Products,
Inc., Calabasas, CA) was used. LNCaP cells were cultured first in RPMI
1640 with 10% cFBS for 5 days and then stimulated with an non-metabolizable
androgen analog R1881 (DUPONT, Boston, MA) 10-8 M for 24 hours (for generating
LNCaP-T library).
LNCaP cells identically treated but without R1881 treatment
served as control (for generating LNCaP-C library). Cells were harvested
at indicated time and polyA+ RNA was double-selected with Fast Track kit
(Invitrogene).
SAGE libraries were generated according to the procedure
provided by Dr. Kinzler (Johns Hopkins University School of Medicine,
Baltimore, MD). Briefly, biotinylated oligo dT primed cDNA was prepared
from five micrograms of polyA+ RNA from treated and control LNCaP cells
and biotinylated cDNA was captured on strepravidin coated magnetic beads
cDNA bound to the magnetic beads were digested by NlaIII followed by ligation
to synthetic linkers containing a site for anchoring enzyme, NlaIII and
a site for tagging enzyme BsmF1. The restriction digestion of ligated
products with BsmF1 resulted in the capture of 10-11 bp sequences termed
as “tags” representing signature sequence of unique cDNAs.
Strategy combining
ligation, PCR, enzymatic digestion and gel purification yielded two tags
linked together termed as “ditags.” Ditags were concatamerized, purified
and cloned in plasmid pZero cloning vector (Invitrogen, CA). The clones
containing concatamers were screened by PCR and sequenced. The sequence
and the occurrence of each of the SAGE tags was determined using the SAGE
software kindly provided by Dr. Kinzler.
AUA Abstract
| 
ARGs and Specific
Genes |
Androgen Up
Regulated Transcripts |
Tables/Figures
