|
| Status |
Public on Jul 16, 2010 |
| Title |
10-87LP3 vs 10-87HP3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
African Green Monkey Kidney Cells
|
| Organism |
Chlorocebus aethiops |
| Characteristics |
cell type: non-tumorigenic
sample type: 10-87LP3
cell line: VERO
|
| Biomaterial provider |
ATCC
|
| Treatment protocol |
The cell lines has been passaged in culture under low density condition. Total RNA (including miRNAs) has been extracted at passage 148.
|
| Growth protocol |
Cells from the World Health Organization (WHO) VERO cell bank 10–87 (ATCC 10–87 VERO cells) were serially passaged in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (DMEM-10) from p134 to p250 by sub-culturing VERO cells before they reached confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using the miRNeasy mini kit according to the manufacturer’s procedures (Qiagen Inc., Valencia, CA).
|
| Label |
Cy3
|
| Label protocol |
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments.
|
| |
|
| Channel 2 |
| Source name |
African Green Monkey Kidney Cells
|
| Organism |
Chlorocebus aethiops |
| Characteristics |
cell type: tumorigenic
sample type: 10-87HP3
cell line: VERO
|
| Biomaterial provider |
Laborator of DNA Virus, FDA
|
| Treatment protocol |
The cell lines has been passed in culture under low density condition and Total RNA (including miRNAs) was extracted at passage 250.
|
| Growth protocol |
Cells from the World Health Organization (WHO) VERO cell bank 10–87 (ATCC 10–87 VERO cells) were serially passaged in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) (DMEM-10) from p134 to p250 by sub-culturing VERO cells before they reached confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using the miRNeasy mini kit according to the manufacturer’s procedures (Qiagen Inc., Valencia, CA).
|
| Label |
Cy5
|
| Label protocol |
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments.
|
| |
|
| |
| Hybridization protocol |
The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 L 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
None
|
| Data processing |
Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression) (2). For two color experiments, the ratio of the two sets of detected signals (log2 transformed, balanced) and p-values of the t-test were calculated; differentially detected signals were those with less than 0.01 p-values.
|
| |
|
| Submission date |
Jul 13, 2010 |
| Last update date |
Jul 15, 2010 |
| Contact name |
Andrew M Lewis |
| E-mail(s) |
andrew.lewis@fda.hhs
|
| Phone |
301-827-0650
|
| Organization name |
Food and Drug Adminstration
|
| Department |
Division of Viral products
|
| Lab |
Labratory of DNA Virus
|
| Street address |
29 Lincoln Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL10649 |
| Series (1) |
| GSE22920 |
Patterns of microRNA Expression in Non-Human Primate |
|
