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Status |
Public on Oct 18, 2010 |
Title |
Ref2.2 (LC) |
Sample type |
RNA |
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Source name |
Total RNA with small fraction
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Organism |
Mus musculus |
Characteristics |
sample: Reference 2 sample type: pool of equal parts RNA from heart, lung and liver microarray barcode: 251911910767
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA containing the small fraction was isolated from each sample using the miRVana miRNA Isolation Kit (Ambion), Streetsville, ON, Canada). Quality was confirmed using the Agilent 2100 Bioanalyzer and RNA Nano 6000 chips.
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Label |
Cy3
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Label protocol |
LC Sciences arrays were performed in one colour by the company at the LC Sciences Headquarters (Houston, TX) using two replicates of each reference pool. Briefly, 1 ug of total reference RNA was extracted with Trizol reagent (Invitrogen) and size fractionated using a YM-100 Microcon filter (Millipore). Small RNAs less than 300 nucleotides in length were extended with a poly(A) tail using poly(A) polymerase. Poly(A) tails were ligated with an oligonucleotide tag for subsequent dye staining.
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Hybridization protocol |
Hybridizations were carried out overnight on µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies) using 100 µL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34°C overnight. Each detection probe on the microfluidic chip consisted of a chemically-modified nucleotide coding segment complementary to target microRNA or other RNA (control sequences), and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining.
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Scan protocol |
Images were acquired using a GenePix 4000B (Molecular Device) laser scanner by LC Sciences. Intensity data were collected using Array-Pro image analysis software (Media Cybernetics) with a scan resolution of 10 microns and PTM between 480 and 540V.
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Description |
Reference RNA was developed from FirstChoice® mouse Total RNA including the small fraction (Catalogue # AM7800-AM7828, Ambion, Streetsville, ON)
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Data processing |
Analysis was conducted on background subtracted signal intensities. Background was determined using a regression-based background mapping method. Spots yielding true signal (i.e., present) had signal intensities higher than 3×(background standard deviation) and standard deviation/signal intensity < 0.5. CV is calculated by (standard deviation)/(signal intensity). Probes repeated multiple times on an array were called present if the signals from at least 50% of the probes were above detection level.
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Submission date |
Dec 28, 2009 |
Last update date |
Oct 18, 2010 |
Contact name |
Andrew Williams |
Organization name |
Health Canada
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Street address |
50 Columbine
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City |
Ottawa |
State/province |
ON |
ZIP/Postal code |
K1A OK9 |
Country |
Canada |
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Platform ID |
GPL8530 |
Series (1) |
GSE19669 |
Cross-platform correlation analysis of global microRNA expression technologies |
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