|
| Status |
Public on Nov 13, 2007 |
| Title |
shma-p53null 48h 2.5 BaP A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
sh251005-p53null 48h DMSO A(sh251005-p53null 48h DMSO A )
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: tumor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were collected and total RNA extracted using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, UK). After addition of lysis buffer (RLT, Qiagen) samples were homogenized using QIAshredders (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (4 ug) was reversed transcribed into cDNA and fluorescently labelled with Cy3 or Cy5 mono-reactive dyes (Amersham Biosciences, UK) using the Invitrogen Indirect cDNA Labelling Kit protocol (Invitrogen, UK) according to the manufacturer's instructions. After labelling, repetitive sequences within the cDNA samples were blocked with 16 ug Human Cot-1 DNA (Invitrogen, UK) to prevent non-specific sequences binding to the cDNA probes.
|
| |
|
| Channel 2 |
| Source name |
sh251005-p53null 48h 2.5 BaP A(sh251005-p53null 48h 2.5 BaP A )
|
| Organism |
Homo sapiens |
| Characteristics |
tissue type: tumor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets were collected and total RNA extracted using the Qiagen RNeasy Mini Kit protocol (RNeasy Mini Handbook, Qiagen, UK). After addition of lysis buffer (RLT, Qiagen) samples were homogenized using QIAshredders (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Total RNA (4 ug) was reversed transcribed into cDNA and fluorescently labelled with Cy3 or Cy5 mono-reactive dyes (Amersham Biosciences, UK) using the Invitrogen Indirect cDNA Labelling Kit protocol (Invitrogen, UK) according to the manufacturer's instructions. After labelling, repetitive sequences within the cDNA samples were blocked with 16 ug Human Cot-1 DNA (Invitrogen, UK) to prevent non-specific sequences binding to the cDNA probes.
|
| |
|
| |
| Hybridization protocol |
Corresponding control and treated samples labelled with different fluorophores were combined in a 50 ul hybridisation mix (19 ul Microarray Hybridisation Solution purchased from GE Healthcare and 50% deionised formamide) and heated at 70°C for 2 min and then at 37°C for 10 min. Sample was pipetted onto a microarray slide and covered with a glass cover slip. A 150ul aliquot of 6 X SSPE was pipetted underneath the slide to prevent dehydration. The hybridisation chamber was sealed and incubated at 42°C for 72 h. Type 7* slides were washed three times in 4 X SSPE, 10 mM EDTA pH 8.0 as above, followed by a 10 s wash in 50% deionised formamide, 6 X SSPE, before a brief rinse in HPLC grade water and drying with nitrogen gas.
|
| Scan protocol |
Scanner: Axon4000A. Voltage: 3.67 3.6. Resolution (micrometers): 10.
|
| Description |
Labelled sample was reduced down to 3 µl using Microcon YM-30 centifugal filters (Millipore, UK). Corresponding control and treated samples labelled with different fluorophores were combined in a 50 µl hybridisation mix (19 µl Microarray Hybridisation Solution purchased from GE Healthcare and 50% deionised formamide) and heated at 70degC for 2 min and then at 37degC for 10 min. Sample was pipetted onto a microarray slide and covered with a glass cover slip. A 150µl aliquot of 6 X SSPE was pipetted underneath the slide to prevent dehydration. The hybridisation chamber was sealed and incubated at 42degC for 72 h.
|
| Data processing |
Regions were used for normalizing samples 1-23. A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead.
|
| |
|
| Submission date |
Nov 08, 2007 |
| Last update date |
Apr 24, 2013 |
| Contact name |
Ian Giddings |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
+44 20 8722 4293
|
| Fax |
+44 20 8722 4141
|
| URL |
http://www.crukdmf.icr.ac.uk
|
| Organization name |
Institute of Cancer Research
|
| Department |
Section of Molecular Carcinogenesis
|
| Lab |
CANCER RESEARCH UK DNA Microarray Facility
|
| Street address |
15 Cotswold Road
|
| City |
Sutton |
| State/province |
Surrey |
| ZIP/Postal code |
SM2 5NG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4348 |
| Series (1) |
| GSE9547 |
p53 status and BaP or BPDE gene expression response |
|
| Data table header descriptions |
| ID_REF |
IMAGE clone ID if the value is numeric. Else, it is an oligo ID. the suffix _ is used for copies |
| VALUE |
log2-transformed normalised ratio (Cy5/Cy3) |
| CH1_MEDIAN |
channel 1 signal intensity median - Cy3 (cyanine 3) |
| CH1_MEAN |
channel 1 signal intensity mean |
| CH1_SD |
channel 1 signal intensity standard deviation |
| CH1_AREA |
Number of pixels used to determine channel 1 signal intensity |
| CH1_BKD_MEDIAN |
channel 1 background signal intensity median |
| CH1_BKD_MEAN |
channel 1 background signal intensity mean |
| CH1_BKD_SD |
channel 1 background signal intensity standard deviation |
| CH1_BKD_AREA |
Number of pixels used to determine channel 1 background signal intensity |
| CH2_MEDIAN |
channel 2 signal intensity median - Cy5 (cyanine 5) |
| CH2_MEAN |
channel 2 signal intensity mean |
| CH2_SD |
channel 2 signal intensity standard deviation |
| CH2_AREA |
Number of pixels used to determine channel 2 signal intensity |
| CH2_BKD_MEDIAN |
channel 2 background signal intensity median |
| CH2_BKD_MEAN |
channel 2 background signal intensity mean |
| CH2_BKD_SD |
channel 2 background signal intensity standard deviation |
| CH2_BKD_AREA |
Number of pixels used to determine channel 2 background signal intensity |
| RATIO |
normalised ratio (Cy5/Cy3) |
