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 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.
Immediately, 120 μl of Pro-Q Diamond™ phosphoprotein/phosphopeptide microarray stain was added to the surface of the microarrays.
Four micro-liters of remaining cRNA not used for microarray hybridization was added to each 25 μl PCR mixture consisting of primers (0.16 μM each) specific to one miR of interest, buffers, salts, and SYBR Green PCR master mix.
For microarray hybridizations, 100 μl of the sample was added to the microarray slide, which was then mounted in an Agilent™ Microarray Hybridization Chamber, within an Agilent G2545A hybridization oven for 17 h at 65°C.
The procedure for testing with microarray is as follows: firstly, 100 μL of PBST was added onto the microarray to wet it.
For hybridization, 1.65 µg of Cy3-labeled cRNA was added on microarray slide for 17 hours using the Hybridization Oven kit procedure provided by Agilent Technologies.
Fifteen µl of this hybridization mixture was added to each microarray and sealed with a coverslip.
Probe was added to the microarray using a lifterslip (Erie Scientific, Portsmouth, NH) and allowed to hybridize in a humidity chamber for 16 hr at 50°C.
