|
| Status |
Public on Jun 13, 2019 |
| Title |
CTRL_4 |
| Sample type |
RNA |
| |
|
| Source name |
CTRL, whole blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: whole blood
gender: female
disease state: CTRL
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A sample of 500 µl of whole blood was mixed with 700 µl of Tri-Pure reagent (ROCHE, USA) well mixed in vórtex and left during 3 minutes at 25°C. Posteriorly 100 ul of Chloroform:Isoamyl alcohol 24:1 were added and centrifuged at 13,000xg for 15 minutes at 4°C. The aqueous phase was then transferred to a pre-filter column of Absolutely-miRNA RNA kit (Agilent) and the extraction continued performed as indicated in the manufacturer’s instructions. RNA was stored at -70°C until use
|
| Label |
Cy3
|
| Label protocol |
total RNA (100 ng) was placed in 1.5 mL microfuge tube. 2 μL of the CIP Master Mix was added to each sample tube and Incubated at 37°C for 30 min. to dephosphorylate the sample.posteriorly 2.8 μL of DMSO was added to each sample and Incubated at 100°C for 10 min. 4.5 μL of the Ligation Master Mix, wich contain Cy3 was added and incubated for 2 hours.
|
| |
|
| Hybridization protocol |
The dried sample was resuspended in 17 μL of nuclease-free water, Hyb Spike-In solution, 10× Gene Expression Blocking Agent and 2× Hi-RPM Hybridization Buffer and were Incubated at 100°C for 5 min. The mixture was hybridized to Agilent miRNA microarray (G4870A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Arrays were scanned using an Agilent Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 5 um . Dye channel was set to Green and Green PMT is set to 100%.
|
| Description |
Gene expression after addition of 1ml RNAlater to human blood 4 ml samples
|
| Data processing |
The generated image files were analyzed using Agilent Feature Extraction Software (v 12.0.1.1). Using default parameters (protocol miRNA_1200_Jun14 and Grid: 070156_D_F_20141006 ) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jun 15, 2018 |
| Last update date |
Jun 14, 2019 |
| Contact name |
María Fernanda Romo García |
| E-mail(s) |
mromog@cinvestav.mx
|
| Phone |
4921236919
|
| Organization name |
IMSS
|
| Street address |
Alameda
|
| City |
Zacatecas |
| State/province |
Zacatecas |
| ZIP/Postal code |
98050 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL25134 |
| Series (1) |
| GSE115885 |
microRNA profiling in different stages of rheumatoid arthritis |
|
