RNA isolation was accomplished using Trizol. The RNA underwent three rounds of linear amplification (Baugh et. al. 2001), the aRNA was reverse-transcribed into cDNA with Superscript III reverse transcriptase for 15h at 42°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.
Label
Cy5
Label protocol
The cDNA from sample and reference was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0. The Cy5 and Cy3 labeled cDNAs were purified separately with a QIAquick PCR purification column. Each labeled sample was analyzed using a spectrophotometer, after which sample and reference were speedvacced, combined, and suspended in 40 µl containing ddH2O, 20 µg of bovine Cot1 DNA, 40 µg oligo dT and 20 ug T7dT24. After denaturation for 3 min at 94°C, reannealing 10 min at 60°C, 2x Hybridization buffer (50% formamide,10x SSC, 02% SDS) is added and mixture is hybridized to the microarray.
reference total RNA isolated by Trizol from EBTr, MDBK, and BL30 cell lines and brain tissue
Extracted molecule
total RNA
Extraction protocol
RNA isolation was accomplished using Trizol. The RNA underwent two rounds of linear amplification (Baugh et. al. 2001) , the aRNA was reverse-transcribed into cDNA with Superscript III reverse transcriptase for 15h at 42°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.
Label
Cy3
Label protocol
The cDNA from sample and reference was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0. The Cy5 and Cy3 labeled cDNAs were purified separately with a QIAquick PCR purification column. Each labeled sample was analyzed using a spectrophotometer, after which sample and reference were speedvacced, combined and suspended in 40 µl containing ddH2O, 20 µg of bovine Cot1 DNA, 40 µg oligo dT and 20 ug T7dT24. After denaturation for 3 min at 94°C, reannealing 10 min at 60°C, 2x Hybridization buffer (50% formamide,10x SSC, 02% SDS) is added and mixture is hybridized to the microarray.
Hybridization protocol
Each labeled sample was combined with a labeled reference,and suspended in a total of 40 µl containing ddH2O, 20 µg of bovine Cot1 DNA, 40 µg oligo dT and 20 ug of T7dT24. After denaturation for 2.5 min at 94°C, reannealing 10 min at 60°C, 2x Hybridization buffer (50% formamide,10x SSC, 02% SDS) is added and the mixture is hybridized to the microarray for 40 hrs at 42 degrees. Arrays were subsequently washed in a series of SSC/SDS washes to remove unbound cDNA. After washing, arrays were spun dry and immediately scanned.
Scan protocol
microarray image was acquired using a GenePix 4000B scanner and analyzed using GenePix Pro 4.0. Fluoresecent images for both dye channels were obtained using a GenePix 4000B (Axon Instruments, Inc., CA) dual-laser confocal scanner and images were processed using GenePix Pro 4.0 (Axon Instruments, Inc., CA).
Description
cDNA from Embryo RNA and the reference standard. Keywords = Embryo Keywords = cow Keywords = AI Keywords = microarray
Data processing
Values are raw log2 tranformed ratios, no normalizations are performed.
