reference total RNA was isolated by Trizol and consists of equal amounts total RNA extracted from EBTr, MDBK, and BL30 cell lines, and brain tissue
Extracted molecule
total RNA
Extraction protocol
RNA isolation was accomplished using Trizol.
Label
Cy5
Label protocol
The reference RNA (10 µg) was annealed with 6 µg random N6, and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 15h at 46°C in the presence of 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 1M NaOH, neutralization with 1M HCl,Tris was removed from the reaction with a Qiagen PCR-column. The cDNA was covalently coupled separately with Cy5-monoreactive dye in 50 mM sodium bicarbonate, pH 9.0. The Cy5-labeled cDNA was purified with a QIAquick PCR purification column. The labeled reference was analyzed using a spectrophotometer to assess incoproration of dyes.
The sample RNA (10 µg) was annealed with 6 µg random N6, and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 15h at 46°C in the presence of 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 1M NaOH, neutralization with 1M HCl,Tris was removed from the reaction with a Qiagen PCR-column. The cDNA was covalently coupled separately with Cy3-monoreactive dye in 50 mM sodium bicarbonate, pH 9.0. The Cy3-labeled cDNA was purified with a QIAquick PCR purification column. The labeled sample was analyzed using a spectrophotometer to assess incoproration of dyes.
Hybridization protocol
Each Cy3-labeled sample was combined with a Cy5-labeled reference, vacuum dried, and resuspended in 40 µl of ddH2O containing 10 µg of bovine Cot1 DNA, 20 µg oligo dT. After denaturation for 3 min at 94°C and reannealing for 10 min at 60°C, 40 µl of 2x hybridization buffer (50% formamide, 10x SSC, 02% SDS) was added and the mixture was hybridized to the microarray for 40 hrs. Microarrays were subsequently washed in a series of SSC/SDS washes (1xSSC/0.2% SDS, 0.1xSS/0.2% SDS, 0.1xSSC)to remove unbound cDNA. After washing, arrays were dried by centrifugation and stored in argon gas until scanning (max. ~1 hr).
Scan protocol
The microarray image was acquired using a GenePix 4000B scanner and analyzed using GenePix Pro 6.0 using the autobalance feature and a saturation threshold of 0.05%.
Description
The cDNA from placentome RNA and the reference standard was coupled with Cy3 or Cy5 fluorescent dye and then co-hybridized to the microarray. Fluoresecent images for both dye channels were obtained using a GenePix 4000B scanner dual-laser confocal scanner and images were processed using GenePix 6.0 (Axon Instruments, Inc., CA). Keywords = placentome Keywords = cow Keywords = term pregnancy Keywords = microarray
Data processing
Values are raw log2 transformed ratios, no normalizations are performed, and only GenePix flagging is performed.
