|
| Status |
Public on Jun 06, 2007 |
| Title |
Healthy donor SN_53_vs_Pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Hs346_SN53
|
| Organism |
Homo sapiens |
| Characteristics |
Blood sample from healthy donor
|
| Treatment protocol |
Blood sample (10 ml) was taken from healthy donor by venipuncture before intervention on the day of surgery, and the first several milliliters were discarded to eliminate skin-plug contamination. Plasma was prepared by centrifugation of peripheral blood at 2500 rpm for 25 minutes and divided into aliquots, which were snap frozen at -80ºC until processing.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Plasma mRNA was obtained from 3 ml of sample using Dynabeads mRNA DIRECT Kit following manufacturer´s instructions. In brief, plasma was incubated with 200 ul of Dynabeads Oligo (dT) for 10 min at room temperature and the mRNA was eluted in 10 mM Tris-HCl. Since low amounts of mRNA were obtained, mRNA was 2-cycle amplified by Eberwine protocol using AminoAllyl MessageAmp aRNA kit (Ambion) following manufacturer instructions.
|
| Label |
Cy3
|
| Label protocol |
aRNA was generated in the second amplification round including Cy3-aminoallyl-UTP for labeling by using CyDye PostLabelling Reactive Dye Pack (GE Healthcare) and following Ambion kit instructions. Labeled aRNA was measured in a Nanodrop1000.
|
| |
|
| Channel 2 |
| Source name |
Normal_Control pool
|
| Organism |
Homo sapiens |
| Characteristics |
Pool of blood samples from 26 healthy donors, obtained at the hematology unit of the hospital.
|
| Treatment protocol |
Plasma was prepared by centrifugation of peripheral blood at 2500 rpm for 25 minutes and snap frozen at -80ºC until processing.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Plasma mRNA was obtained from 3 ml of sample using Dynabeads mRNA DIRECT Kit following manufacturer´s instructions. In brief, plasma was incubated with 200 ul of Dynabeads Oligo (dT) for 10 min at room temperature and the mRNA was eluted in 10 mM Tris-HCl. Since low amounts of mRNA were obtained, mRNA was 2-cycle amplified by Eberwine protocol using AminoAllyl MessageAmp aRNA kit (Ambion) following manufacturer instructions.
|
| Label |
Cy5
|
| Label protocol |
aRNA was generated in the second amplification round including Cy5-aminoallyl-UTP for labeling by using CyDye PostLabelling Reactive Dye Pack (GE Healthcare) and following Ambion kit instructions. Labeled aRNA was measured in a Nanodrop1000.
|
| |
|
| |
| Hybridization protocol |
Prehybridization was performed at 42ºC for 30-45 minutes in 6X SSC, 0.5% SDS and 1% BSA. Slides were rinsed five times with destilled water. Cy5 and Cy3 amplified RNA (aRNA) probes were mixed (30-50 pmol of each label) with 10 µg of PolyA (Sigma) and 1 µg of Cot-DNA (Invitrogen) in a final volume of 40 µl of hybridization buffer (50% formamide, 6X SSC, 0.5% SDS, 5X Denhardt’s). The probe was denatured at 95ºC for 5 minutes and applied to the slide using a coverslip. Slides were then incubated at 42ºC for 16h in hybridization chambers (Array-It). After incubation, slides were washed twice with 0.1X SSC, 0.1% SDS for 5 minutes each, three times with 0.1X SSC for 5 minutes and finally in distilled water for 10 seconds. Slides were dried by centrifugation at 563g for 1 minute.
|
| Scan protocol |
Images from Cy3 and Cy5 channels were equilibrated and captured with an Axon 4000B scanner (Axon Instruments) and spots quantified using GenePix 5.1 software (Axon). GPR files were then included in almaZen database software (Bioalma). Signals were background subtracted, and normalized by total intensity and lowess_pritn_tip_group.
|
| Description |
Informed consent was obtained from all participants following an explanation of the nature of the study, as approved by the research ethics board of our hospital.
|
| Data processing |
GPR files with raw data were introduced in almaZen database software (Bioalma). Signals were background subtracted, and normalized by total intensity and Print_tip_group_Lowess.
|
| |
|
| Submission date |
Jun 05, 2006 |
| Last update date |
Jun 06, 2006 |
| Contact name |
Luis LOMBARDIA |
| E-mail(s) |
[email protected]
|
| Organization name |
CNIO (Spanish National Cancer Center)
|
| Department |
Molecualr Pathology Programme
|
| Lab |
Molecular Diagnostics Unit
|
| Street address |
Melchor Fernandez Almagro, 3
|
| City |
MADRID |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL3014 |
| Series (1) |
| GSE4988 |
Gene expression profiling of circulating plasma RNA from colorectal cancer patients |
|
