Total RNA was extracted from a pooled sample of approximate 60,000 prism stage urchin larvae. Total RNA was isolated using TRIzol® Reagent following manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA was then processed through an additional column clean-up step to remove tRNA and small-sized RNA degradation products.
Label
Alexafluor 647
Label protocol
Indirect coupling of dye following cDNA synthesis with amino-allyl dUTP. cDNA was incubated with 1 μL of either Alexafluor 555 or 647 dye for 1h at room temperature in the dark.
Total RNA was extracted from a pooled sample of approximate 60,000 prism stage urchin larvae. Total RNA was isolated using TRIzol® Reagent following manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA was then processed through an additional column clean-up step to remove tRNA and small-sized RNA degradation products.
Label
Alexafluor 555
Label protocol
Indirect coupling of dye following cDNA synthesis with amino-allyl dUTP. cDNA was incubated with 1 μL of either Alexafluor 555 or 647 dye for 1h at room temperature in the dark.
Hybridization protocol
Samples were hybridized following Agilent’s Protocol for Two-Color Microarray-Based Gene Expression Analysis (version 5.5, pages 24-35). The cDNA samples were quantified using a NanoDrop 1000 UV/visible spectrophotometer (Thermo Scientific, Wilmington, DE, USA) to ensure high quality cDNA synthesis and dye incorporation before continuing on to the slide hybridization. If the cDNA yield of a particular sample was <600 ηg and the specific activity was <8.0 ρmol dye per μg cDNA, cDNA synthesis and labeling was repeated. Prior to slide hybridization, cDNA samples were fragmented following Agilent’s Gene Expression Hybrization Kit for 4x44K slides. Briefly, 600ηg of Alexafluor 555-labeled cDNA, 600ηg of Alexafluor 647-labeled cDNA, 11 μL of 10X Blocking Agent and 2.2 μL of 25X Fragmentation Buffer were mixed to a total volume of 55 μL with nuclease-free water and incubated at 60°C for 30 min. To each array mixture 55 μL of 2X GE Hybridization Buffer HI-RPM was added and then the sample was applied to the microarray slides. Hybridizations were conducted overnight (~17 h) at 65°C in Agilent Microarray Hybridization Chambers with the hybridization oven rotisserie set at 10rpm. Following hybridization, the slides were washed in Agilent’s Gene Expression Wash Buffers following manufacturer’s instructions.
Scan protocol
The slides were scanned at 5μm on a GenePix 4000B microarray scanner (Axon Instruments, Molecular Devices, Sunnyvale, CA, USA). Data from the microarrays were extracted using GenePix Pro 4.0 software (Axon Instruments).
Description
N/A
Data processing
Normalization and data analysis were completed using R (R Development Core Team 2008) with the limma software package (Smyth 2005). To avoid violating assumptions of the number or degree of symmetry of differentially expressed genes, a global normalization for dye-bias was applied on a probe-by-probe basis, after averaging over log-ratios from replicate probes within arrays, by including a dye-effect in the linear model.
