|
| Status |
Public on Mar 25, 2008 |
| Title |
P12_L2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Renal tumor tissue from patient 12
|
| Organism |
Homo sapiens |
| Characteristics |
Renal tumor tissue from patient 12; Histological pattern: clear cell
|
| Biomaterial provider |
Instituto Nacional do Cancer (INCA) - Rio de Janeiro - Brazil
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from tissue samples (approximately 200 mg) with Trizol reagent (Invitrogen), quantified by UV spectrophotometry (Nanodrop ND1000) and analysed for integrity by inspection of 18S/28S ribosomal RNA ratios following separation by gel electrophoresis.
|
| Label |
Cy5
|
| Label protocol |
For each patient, aliquots containing 20 ug of total RNA from tumor or adjacent non-tumor tissue were labeled in parallel with either Cy3- or Cy5-derivatized deoxyribonucleotides (CyScribe first-strand cDNA labeling kit; GE Healthcare) using a mixture of oligo-dT and 9-mer random oligonucleotides as primers.
|
| |
|
| Channel 2 |
| Source name |
Adjacent non-tumor renal tissue from patient 12
|
| Organism |
Homo sapiens |
| Characteristics |
Adjacent non-tumor renal tissue from patient 12
|
| Biomaterial provider |
Instituto Nacional do Cancer (INCA) - Rio de Janeiro - Brazil
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from tissue samples (approximately 200 mg) with Trizol reagent (Invitrogen), quantified by UV spectrophotometry (Nanodrop ND1000) and analysed for integrity by inspection of 18S/28S ribosomal RNA ratios following separation by gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
For each patient, aliquots containing 20 ug of total RNA from tumor or adjacent non-tumor tissue were labeled in parallel with either Cy3- or Cy5-derivatized deoxyribonucleotides (CyScribe first-strand cDNA labeling kit; GE Healthcare) using a mixture of oligo-dT and 9-mer random oligonucleotides as primers.
|
| |
|
| |
| Hybridization protocol |
Labeled targets were ressuspended in 250 (l of 1 x hybridization buffer (25% formamide, 12.5% of proprietary Microarray Hybridization Buffer Version 2 from Amersham Biosciences cat. RPK0325) denatured for 2 min at 92 ºC and centrifuged at 13,000 rpm for 5 min. All slide processing steps (blocking, hybridization, washing) were carried out on an automated slide processor (ASP) from Amersham Biosciences. The slides were incubated for 16h at 42ºC and subsequently washed at room temperature in 1xSSC/0.2%SDS for 5min, 0.1xSSC/0.2%SDS for 5 min and in 0.1XSSC for 3 min. After the washing steps, slides were flushed with isopropanol and dried with an air flush at 42ºC.
|
| Scan protocol |
Images were obtained from each channel by laser scanning using GenePix 4000B microarray scanner (Axon Instruments). Slides were scanned with the following parameters: scanning resolution: 10 -(m pixel; excitation wave length: 633 nm (Cy5-labeled targets); emission filter: 675 nm (Cy5-labeled targets); PMT voltage: 600 volts (Cy5-labeled targets).
|
| Description |
Hybridization included external mRNA spikes for quality control of the experiment (Lucidea Microarray ScoreCard). Raw images were analyzed using the ArrayVision software (Version 8.0, Imaging Research Inc.). An array template (or grid) was first automatically aligned to locate the position of each spot in the array, and subsequently manually adjusted to obtain the best possible alignment.
|
| Data processing |
We employed in our analyses the Artifact-Removed density value (ARM) calculated for each spot by ArrayVision. The ARM value represents the average of all the pixels remaining in the spot, after first removing pixels with density values that exceed four median absolute deviations (MADs) from the median. The ARM density for each spot was subtracted by the median background surrounding the spot (distance of 2 pixels with 3 widths of pixels).The raw data of each spot was compared to the value of hibridization background using the Lucidea Microarray Scorecard to determine which spots have a signal above or below the detection limit of each hybridization, where the background was given by the signal measured on a negative control (plant cDNA). If their signal were at least 3 standard deviations above the average signal of the negative control, spots were considered expressed in tissues. We decided to use the mean intensity signal (40% trimmed) of each dataset for normalization between experiments.
|
| |
|
| Submission date |
Mar 26, 2007 |
| Last update date |
Mar 25, 2008 |
| Contact name |
Glauber Costa Brito |
| E-mail(s) |
[email protected]
|
| URL |
http://verjo2.iq.usp.br
|
| Organization name |
Universidade de Sao Paulo
|
| Department |
Instituto de Quimica - Dep Bioquimica
|
| Lab |
Expressao Genica em Eucariotos
|
| Street address |
Av. Prof. Lineu Prestes, 748 - sala 1.200
|
| City |
Sao Paulo |
| State/province |
Sao paulo |
| ZIP/Postal code |
05508-000 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL3985 |
| Series (1) |
| GSE7367 |
Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma |
|
