Fixed concentration of short fluorescent oligonucleotide control sequences ('spikes'), complementary to the control oligonucleotides on microarray, were added to the final 80 μl hybridization solution for normalization purposes.
Positive control transcripts, i.e. spike-in transcripts complementary to the standard control probes on microarray, were added to high quality total RNA samples and common biological reference (RNA Integrity Number [RIN] > 9.6) using the Agilent RNA Spike-In Kit for monitoring the microarray workflow.
The best quality probes from the commercially available Agilent transcriptome-wide rabbit microarray was added, resulting in a microarray with 62, 976 probes including positive and negative controls.
The microarrays were added to preheated (42°C) pre-hybridization buffer [20% Formamide (v/v), 5× Denhardt's, 6× SSC, 0.1% SDS (w/v), 25 µg/ml tRNA] and incubated at 42°C for 45 min, shaking occasionally.
For the RNA-Seq assembled transcripts, re-normalized microarray expression data were added to the TetraFGD to help researchers get more accurate expression information, and the result gives the summary information, sequence, as well as the expression profile.
The two labelled aRNA were added to Microarray Hybridization Buffer Version 2 (GE HealthCare, St Catharines, Ontario) in a final concentration of 50% formamide, denaturated at 95° C for three minutes and applied to the microarrays in individual chambers of an automated slide processor (GE HealthCare, St Catharines, Ontario).
In the microarray assay, two other multiparous cows were added to each period, and mixed RNA samples were made.
Hybridisation solutions were added to each microarray in a Hybridisation Chamber (Agilent) and incubated at 50°C for 18 hr.
Three undifferentiated/differentiated cell couples were used for the microarray study and three new couples were added for real-time PCR analysis.
Both the 3DNA capture reagents with Cy3 and Cy5, in a SDS-based hybridization buffer, were added to the microarray slide under a supported glass coverslip.
In addition to the 7 matched pairs used for the microarray analysis, 6 extra matched pairs were added to enlarge the cohort to 13 matched pairs.
