|
| Status |
Public on Jul 18, 2007 |
| Title |
Bovine Control Sample # 1 |
| Sample type |
RNA |
| |
|
| Source name |
The bovine adrenal glomerulosa cells were isolated from male and female near term fetal bovine calves.
|
| Organism |
Bos taurus |
| Characteristics |
The file represents the control sample used for this experiment.
|
| Biomaterial provider |
Dr Wendy B Bollag Laboratory
|
| Treatment protocol |
Cells were then rinsed with bicarbonate-buffered Kreb's Ringer solution containing 2.5 mM sodium acetate (KRB+) and incubated for 30 min (in 5 % CO2) in KRB+ before addition of KRB+ without or with 10 nM AngII. After 1 hour incubation medium was aspirated and cells were frozen until processing to obtain RNA.
|
| Growth protocol |
The bovine adrenal glomerulosa cells were isolated from male and female near term fetal bovine calves and cultured overnight in Falcon Primaria dishes (Becton Dickinson labware, Lincoln Park, NJ) in a DMEM/Ham’s F12 medium (1:1) containing: 10 % horse serum (vol/vol), 2 % fetal bovine serum (vol/vol), 100 μM ascorbate, 1.2 µM alpha-tocopherol, 0.05 μM Na2SeO3, 50 µM butylated hydroxyanisole, 5 µM metyrapone, 100 U/mL penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells plates using Trizol (Invitrogen) according to the manufacturer’s directions.
|
| Label |
ENZO LABELING KIT
|
| Label protocol |
Mix 20ul of cDNA from above (from 5ug of RNA), Xul Water (Final volume should be 40 ul), 3ul of 10X HY, 3ul Labeled ribonucleotides, 3ul 10XDTT, 3ul 10XRNase Inhibitor, 1.5ul 20X T7 RNA polymerase. Add above at RT to tube in order listed. Mix by centrifugation 5 sec. Place at 37 oC for 5hrs. Remove and mix about every 30-45 minutes.Place in freezer at –20 overnight
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| |
|
| Hybridization protocol |
HYBRIDIZATION
Sample +
Fragmented cRNA 40 40
Control oligo B2 3.9 3.9
Control cRNA from Affy 12 12
Herring Sperm DNA 2.4 2.4
Acetylated BSA 2.4 2.4
2XMES Hybrid buffer 120 120
Water to final volume 59.3 59.3
The control RNA must be heated at 65o C for 5 minutes before adding to mix (use Affymetrix kit)
Warm chips to room temperature
Set up the experiment in GCOS
Experiment name
Project- name of the PI
Sample name could be same as experiment.
Read the bar code on chip and all the other information is filled up.
Add 1X MES to chip and incubate at 45o C for 10 minutes
Heat hybridization cocktail to 99o C for 5 min and then 45o C for 5 minutes
Spin for 5 min
Remove buffer solution from the chip and add 200ul of hybridization cocktail.
Hybridize for 16 hours
Prepare buffer A and B
Non-stringent Wash Buffer A
For 1000 mL
300 ml of 20X SSPE
1.0 mL of 10% Tween-20
698 water
Filter through 0.2um filter
Stringent Wash Buffer B
For 1000 ml
83.3 mL of 12X MES stock buffer (refer chapter 5 of the manual to prepare 12X MES)
5.2 mL of 5M NaCl
1.0ml of 10%tween 20
910.5 mL of water
Filter through 0.2um filter
Next day
Prepare the stains and antibody solution for x number of samples according to the chart on the desk.
Switch on fuidics and scanner and then click on GCOS.
Change water bottles on fuidics to buffer A and B at the designated places.
Prime fuidics by choosing
Prime_450 in the protocol dropdown list
Remove cocktail from the chip and fill with buffer A.
In the Fluidics Station dialog box on the workstation, select the correct experiment in the dropdown Experiment list.
In the protocol dropdown list, select the specific satin protocol which is in most cases FlexGE WS3.
Choose run in the fluidics Station dialog box to begin the washing and staining and follow the instruction on the LCD window of the fuidics.
When the wash is complete the LCD window will display EJECT CARTIDGE.
Remove the cartridge and follow the instruction on LCD window again.
Run the shutdown protocol replacing water bottles with buffer.
There should not be any air bubble in the chip.
|
| Scan protocol |
The scanner is also controlled by GCOS. Put the chips in the scanner. You can scan as many as 48 chips at the same time. Click the start icon in GCOS and the follow the direction there on. After all the chips are scanned, data can be analyzed.
Analysis
Open the data files. Check the grid corners by pressing F5, F6, F7 and F8. Then press the analyze icon. Choose target signal 150 then proceed for analysis. Analyzed data is presented as excel spreadsheet and present as .CHP file. Check for GAPDH5’/3’ signal intensity. The ratio should be less then 4.
|
| Description |
Quality control steps taken:
Sample from treated cells were compared to untreated (control) using different arrays. 3 replicates were used for human array. No dye-swap was used in any experiment.
|
| Data processing |
Software: GeneSpring (Agilent)
Data (.chip files) were inserted into the program and RMA normalization was selected. Then we used a protocol on GeneSpring to create an experiment. The parameters were: Basal versus Angiotensin II
Normalization order: Data Transformation: set measurements less than 0.01 to 0.01; Normalization to the 50th percentile
Error model for one-color data was selected
Base error model on deviation from one selected
Experiment interpretation:
Analysis mode: ration (signal/control); Use measurements flagged: flags present in both arrays.
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| |
|
| Submission date |
Jul 11, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Edson da Fonseca Nogueira |
| E-mail(s) |
enogueira@students.mcg.edu
|
| Phone |
706-721-8779
|
| Fax |
706721-8360
|
| Organization name |
Medical College of Georgia
|
| Department |
Physiology
|
| Lab |
Dr William E Rainey
|
| Street address |
1120 15th Street, CA3091
|
| City |
Augusta |
| State/province |
GA |
| ZIP/Postal code |
30912 |
| Country |
USA |
| |
|
| Platform ID |
GPL2112 |
| Series (1) |
| GSE8442 |
Profiling of Angiotensin II Rapid Response Genes in Human, Bovine, and Rat Adrenocortical Cells. |
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