|
| Status |
Public on Apr 10, 2006 |
| Title |
B27LPSvsCLPS GA8 RG |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
B27 U937 LPS Treated Cy5
|
| Organism |
Homo sapiens |
| Characteristics |
HLA-B27 stable U937 transfectants LPS treated Cy5 labelled
|
| Treatment protocol |
U937 cells were differentiated with PMA (Sigma, U.K) (10ng/ml) for 24 hours followed by 4 hour culture with or without 500ng/ml LPS.
|
| Growth protocol |
Transfectants were cultured in RPMI 1640 (Invitrogen, U.K) supplemented with 10% fetal bovine serum (Sigma U.K), 2mM glutamine and 20mM Hepes in a humidified 5% CO2 atmosphere at 37oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from U937 cell lines using RNA STAT60TM (Tel-Test “B” Inc) and further purified using RNeasyTM columns (Qiagen).
|
| Label |
dCTP-Cy5
|
| Label protocol |
cDNA was amplified from total RNA using template-switching PCR and labeled with Cy3 or Cy5 dyes as previously described (Petalidis et al, 2003, NAR). The amplified cDNA was purified on an AutoSeq G-50 column (Amersham Biosciences) according to the supplied protocol. 1μg of the amplified cDNA was labeled using the BioPrimeTM DNA labelling system (Invitrogen) in a 100μl reaction as described by the manufacturer, CyDyeTM5-dCTP or CyDyeTM3-dCTP (Amersham Biosciences). The labelled products were purified on an AutoSeq G-50 columns, the Cy5 and Cy3 samples were then pooled and ethanol precipitated.
|
| |
|
| Channel 2 |
| Source name |
C U937 LPS Treated Cy3
|
| Organism |
Homo sapiens |
| Characteristics |
pRc vector stable U937 transfectants LPS Treated Cy3 labelled
|
| Treatment protocol |
U937 cells were differentiated with PMA (Sigma, U.K) (10ng/ml) for 24 hours followed by 4 hour culture with or without 500ng/ml LPS.
|
| Growth protocol |
Transfectants were cultured in RPMI 1640 (Invitrogen, U.K) supplemented with 10% fetal bovine serum (Sigma U.K), 2mM glutamine and 20mM Hepes in a humidified 5% CO2 atmosphere at 37oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from U937 cell lines using RNA STAT60TM (Tel-Test “B” Inc) and further purified using RNeasyTM columns (Qiagen).
|
| Label |
dCTP-Cy3
|
| Label protocol |
cDNA was amplified from total RNA using template-switching PCR and labeled with Cy3 or Cy5 dyes as previously described (Petalidis et al, 2003, NAR). The amplified cDNA was purified on an AutoSeq G-50 column (Amersham Biosciences) according to the supplied protocol. 1μg of the amplified cDNA was labeled using the BioPrimeTM DNA labelling system (Invitrogen) in a 100μl reaction as described by the manufacturer, CyDyeTM5-dCTP or CyDyeTM3-dCTP (Amersham Biosciences). The labelled products were purified on an AutoSeq G-50 columns, the Cy5 and Cy3 samples were then pooled and ethanol precipitated.
|
| |
|
| |
| Hybridization protocol |
For each set of conditions tested, duplicate and dye-swap hybridizations were performed. Labeled targets were resuspended in 30μl of hybridization buffer (40% formamide, 5x SSC, 5x Denhardt's solution, 1 mM sodium pyrophosphate, 50 mM Tris pH 7.4, 0.1% SDS) together with 2μg human Cot1 DNA (Invitrogen), denatured at 95°C for 5 min, incubated at 50°C for 5 min and then centrifuged at 13,000 r.p.m. for 5 min before being applied to the arrays. Hybridizations were performed under a coverslip at 50°C in a humidified chamber for 16 h. Following hybridization, slides were washed twice in 2x SSC for 10 min, twice in 0.1x SSC/0.1% SDS for 5 min and finally twice in 0.1x SSC for 5 min; all washes were performed at RT. After washing, slides were dried by centrifugation at 2000g for 3 min.
|
| Scan protocol |
Arrays were scanned on an Agilent G2565 scanner according to manufacturer’s instructions. Raw image data were extracted using BlueFuse (BlueGnome Ltd., Cambridge UK).
|
| Description |
Data were imported into GeneSpringTM 7.2 (Silicon Genetics) for analysis and normalization was performed using the Loess algorithm. Identification of genes with ratios statistically significantly different from 1 was performed using a t-test at the 95% confidence level.
|
| Data processing |
Raw image data were extracted using BlueFuse (BlueGnome Ltd., Cambridge UK).
|
| |
|
| Submission date |
Mar 30, 2006 |
| Last update date |
Apr 03, 2006 |
| Contact name |
Giles Yeo |
| E-mail(s) |
gshy2@cam.ac.uk
|
| Phone |
+44 (0)1223762620
|
| Fax |
+44 (0) 1223762323
|
| Organization name |
University of Cambridge
|
| Department |
Clinical Biochemistry
|
| Street address |
CIMR, Addenbrookes Hospital
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 2XY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL3348 |
| Series (1) |
| GSE4579 |
B27 modulation of LPS response |
|
