other: Composed of total RNA from nine human tissues or cell lines: placental tissue, adult liver tissue, adult brain tissue, adult heart tissue, adult breast tissue, adult colon tissue, adult testis tissue, fetal brain tissue, testis cell line.
Biomaterial provider
Agilent Technologies, Cedar Creek, TX
Extracted molecule
total RNA
Extraction protocol
Agilent RNA Extraction Other: not available
Label
cy3
Label protocol
Cy3 Labeling Protocol Label amount: 500 ng Other: Human tissue RNA (Agilent, Santa Clara, CA) was amplified and labeled with Cy3-CTP using Agilent Quick Amp Labeling kit (Agilent, Santa Clara, CA).
tissue: Bone Marrow cell type: Bone Marrow Stromal Cell (BMSC) individual: 09FC45
Treatment protocol
In-vitro treatment: Marrow was collected from the posterior iliac crest of 7 healthy donors. A total of 1.5 to 4.5 mL of marrow was collected in Bone Marrow Prep Syringes (Pharmacy Department, NIH, Bethesda, MD) and then washed with Hank's Balanced Salt Solution (Lonza, Walkersville, MD) (HBSS). A single cell suspension was made with BMSC culture media (BMSC CM) [alpha MEM with 2 mM glutamine (Lonza), supplemented with 20% lot-selected FBS (Hyclone, Thermo Fischer Scientific, Waltham, MA) and 10 ug/ml Gentamicin] and plated at a density of 2 x 10(5)/cm(2) in T-75 flasks (Culture flasks, canted neck, polystyrene, Corning Life Sciences, Corning NY) and were incubated at 37 C in 5% CO2. Non adherent cells were removed after 24 hours and the media was changed. Thereafter, the media was changed every three days until the colonies reach 70-80% confluence and were harvested, which generally was on day 13 of culture. The primary BMSCs were washed with 10mL HBSS twice and incubated with 5mL recombinant human trypsin (TrypLE Express, Invitrogen, Life Technologies, Grand Island, NY) for 10 minutes; and then neutralized with 5mL of BMSC CM, rinsed with 5ml HBSS and centrifuged at 406 x g for 10 minutes. The cell number was counted and viability was assessed by Trypan Blue exclusion method. The cells harvested at this stage were designated as Passage 1, and serial passage numbers were designated thereafter. After passage 1, the BMSCs were seeded in T-75 flasks at a density of 3000 cells per cm2 and incubated 37 C in 5% CO2 until the cells reach 70-80% confluence, during which time the culture medium was changed every three days. The cells were harvested as described above. The number of BMSCs at every passage was manually counted using a C-Chip Disposable Hemacytometers (Incyto, Chonhung, Korea) and viability was assessed by the Trypan Blue exclusion method. The Population Doubling for each passage was calculated using the below equation: N=[log10(NH)-log10(N1)]/log10(2) where N = population doublings, N(H) = cell harvest number, and N(I) = plating cell number. Cumulative population doublings (PD) were calculated in relation to the cell numbers at the first passage. Population doubling time (PDT) was calculated by the below equation on each passage, PDT= time of culture (hrs)/N, where N = population doubling.
Extracted molecule
total RNA
Extraction protocol
Qiagen miRNeasy Mini Kit Protocol Other: The total RNA was extracted using columns in Qiagen miRNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. RNA integrity was assessed by the presence of 28S and 18S bands using Agilent bioanalyzer and quantified using Nano Drop 2000 (Thermo Scientific, Wilmington, DE) with A260/A280 ratios > 2.0 after dilution in DEPC water.
Label
cy5
Label protocol
Cy5 Labeling Protocol Label amount: 500 ng Other: Total RNA (0.5 μg) was amplified and labeled with Cy5-CTP using Quick Amp Labeling kit (Agilent, Santa Clara, CA).
Hybridization protocol
Agilent Hybridization Protocol Solution: 2x GEx Hybridization Buffer HI-RPM Blocking agent: 10x Blocking Agent Wash procedure: The hybridization gaskets were dissembled in GE Wash Buffer 1, washed in GE Wash Buffer 1 for 1 min at room temperature, and then washed in GE Wash Buffer 2 for 1 min at 37 C. Qty. of labeled target: 750 ng Time: 16 hours Other: 825 ng of amplified cRNA were pooled, fragmented and then hybridized on 4x44K microarrays (Agilent) for 17 hours at 65 C.
BRB ArrayTool Data Processing Calculation Method: Analyzed using BRBArrayTools developed by the Biometric Research Branch, National Cancer Institute (http://linus.nci.nih.gov/BRB-ArrayTools.html ). Spots with signal intensity less than 20 in both channels were filtered out. If the signal intensity was less than 20 in one channel only, it was set to 20 in computing the log-ratio value. Global median normalization was performed on the filtered data by subtracting out the median log-ratio for each array. FeatureExtractor_Version: 9.5.1.1 Protocol_Name: GE2-v5_95_Feb07 (Read Only)
