Microarray processing

Microarray processing of all endometriosis serum samples (both original and controls) was carried out according to the manufacturer’s procedure outlined in the Agilent Technologies miRNA Microarray System with miRNA Complete Labelling and Hyb Kit protocol (Version 2.4).

2.6.4.1 Spike-in solution preparation

FIGURE 2-4

Spike in solution preparation⁴⁸⁵

Agilent’s miRNA Spike-In kit (p/n 5190-1934) consists of two miRNA spike-in solutions for process control. Their use is to help distinguish significant biological data from processing issues. The labelling spike-in solution was spiked into the labelling reaction while the Hyb spike-in solution was spiked into the hybridization reaction. Components in the kit were thawed and spun for adequate mixing (Figure 2-4).

Five tubes were labelled as ‘1st Dilution Labelling Spike-In’ and an additional 5 tubes ‘1st Dilution Hyb Spike-In’. 198μL of the dilution buffer was added to each of the 10 tubes and 2μL of the labelling spike-in solution was added to each of the 5 tubes that were labelled ‘1st Dilution Labellspike-ing Spike-In’. 2μL of the Hyb In solution were added to each of the 5 tubes that were labelled ‘1st Dilution Hyb Spike-In’ tubes. Tubes were briefly spun and mixed well. One aliquot of the ‘1st Dilution Labelling Spike-Spike-In’

and ‘1st Dilution Hyb SpikeIn’ were used at a time and the other aliquots were stored in freezers at -80°C.

Four new tubes were labelled as follows: ‘2nd Dilution Labelling Spike-In’, ‘3rd Dilution Labelling Spike-In’, ‘2nd Dilution Hyb Spike-In’, ‘3rd Dilution Hyb Spike-In’ and 198µL of nuclease-free water were added into each of the tubes. To make the second dilution solutions, 2μL from the ‘1st Dilution Labelling Spike-In’ tube were added into the ‘2nd Dilution Labelling Spike-In’ tube. 2μL from the ‘1st Dilution Hyb Spike-In’ tube were added into the ‘2nd Dilution Hyb Spike-In’ tube. Samples were mixed and spun briefly.

To create the third dilution solution 2μL from the ‘2nd Dilution Labelling Spike-In’ tube were added into the ‘3rd Dilution Labelling Spike-In’ tube. 2μL from the ‘2nd Dilution Hyb Spike-In’ tube were added into the ‘3rd Dilution Hyb Spike-In’ tube.

2.6.4.2 Sample labelling and hybridization

Agilent’s miRNA Complete Labelling and Hyb Kit (p/n 5190-0456) generates fluorescently-labelled miRNA with a sample input of 100ng of total RNA. This method involves the ligation of one Cyanine 3-pCp molecule to the 3' end of an RNA molecule.

2.6.4.3 Dephosphorylation

The total RNA sample was diluted to 50ng/μL in DNase/RNase-free water. 2μL (100ng) of the diluted total RNA was placed in a 1.5mL microcentrifuge tube and maintained on ice. The calf intestinal alkaline phosphatase (CIP) master mix was prepared from the following protocol (Table 2-3).

TABLE 2-3

CIP Master Mix with labelling spike-in solution

Components Volume (μL) per

reaction

Volume (μL) per 9 reactions

10X calf intestinal phosphatase buffer 0.4 3.6

Labelling spike-in 1.1 9.9

Calf intestinal phosphatase 0.5 4.5

Total volume 2.0 18.0

Protocol for preparation of calf intestinal alkaline phosphatase (CIP) master mix– Aligent miRNA Microarray system with miRNA complete labelling and hybridisation kit

2μL of CIP master mix was added to each sample tube for a total reaction volume of 4μL.

Dephosphorylation was achieved by incubation at 37°C in a circulating water bath. Denaturation of samples was achieved by adding 2.8μL of 100% DMSO to each sample and incubating them at 100°C in a circulating water bath for 10 minutes. Samples were then transferred to an iced water bath after incubation to prevent re-annealing of RNA.

2.6.4.4 Ligation step

Ligation was achieved by the addition of 4.5μL of the ligation master mix to each sample tube for a total reaction volume of 11.3μL. Samples were gently mixed and incubated at 16°C in a circulating water bath for 2 hours. The samples were then dried using a vacuum concentrator at 45 to 55°C. Samples were deemed dry when pellets were seen not to move or spread on tube flicking. Completely drying the sample ensured that there was no residual DMSO which would adversely affect the hybridization results.

2.6.4.5 Hybridisation step

For hybridisation, a water bath was prepared at 100°C. The dried sample was re-suspended in 17μL of nuclease-free water. The 10X blocking agent was reconstituted in 125μL of nuclease-free water and 4.5μL of the 10X blocking agent was added to each sample. 22.5μL of 2X Hi-RPM hybridization buffer was added to each sample for a total of 45μL. The sample was mixed well on a vortex and was incubated at 100ºC for 5 minutes. It was then immediately transferred to a water bath for 5 minutes. Samples were then quickly spun to collect condensation at the base of the tube.

2.6.4.6 Preparation of the hybridization assembly

The slides were successfully scanned (Agilent Scanner C, at 3 microns) and feature extracted (using Agilent Feature Extraction software, version 10.7.3).

For instructions on how to load slides, assembly and disassembly of chambers, the Agilent Microarray Hybridization Chamber User Guide was used (G2534-90002). A clean gasket slide was added into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. It was ensured that the gasket slide was flush with the chamber base and not ajar. For Agilent scanning and feature extraction, the slides were positioned so that the barcode label was on the left and samples were loaded left to right. Slow dispension of all of the volume of the hybridization sample into the central gasket well was performed. Care was taken to avoid bubble trapping. An array

‘active side’ was slowly placed down onto the SureHyb gasket slide so that the ‘Agilent’-labelled barcode was facing down and the numeric barcode was facing up. Verification that the sandwich-pair was properly aligned was ascertained. The SureHyb chamber cover was placed onto the sandwiched slides and the clamp assembly was slid and tightened onto both pieces. The assembled chamber was vertically rotated to wet the gasket and assess the mobility of the bubbles. The assembled slide chamber was placed in rotisserie in a hybridisation oven set to 55°C at 20 rpm for 20 hours.

2.6.4.7 Microarray wash

2mL of the provided 10% Triton X-102 were pipetted into the wash buffer 1 and 2 respectively in the cubitainer. The caps of the cubitainer were replaced and the solutions were mixed. 1000mL of Gene Expression Wash Buffer 2 was added directly into a sterile 1000mL bottle and placed in a 37°C water bath the night before the washing of the arrays. A slide-staining dish (dish 3) was placed into a 1.5L glass dish three-fourths filled with water. This was warmed to 37°C by storing overnight in an incubator set to 37°C. All equipment was washed copiously with Mill-Q water to remove contaminants.

Slide-staining dish 1 was filled with gene expression wash buffer 1 at room temperature. A slide rack was placed into slide-staining dish 2 and a magnetic stir bar was added. Slide-staining dish 2 was filled with enough Gene Expression Wash Buffer 1 at room temperature to cover the slide rack and this dish was placed on a magnetic stir plate. The pre-warmed 1.5L glass dish was filled with water and contained slide-staining dish 3 on a magnetic stir plate with the heating element. Slide-staining dish 3 was filled to approximately three-fourths capacity with gene expression wash buffer 2 warmed to 37°C. A magnetic stir bar was added and the heating element maintained the gene expression wash buffer 2 at 37°C. A thermometer was used to check the temperature. One hybridisation chamber was removed from the incubator. The time and presence of any bubbles was recorded. Any significant loss of hybridisation volume was noted.

2.6.4.8 Hybridisation chamber disassembly

The hybridisation chamber assembly was placed on a flat surface and the thumbscrew was loosened turning counter-clockwise. The clamp assembly was slid off and the chamber cover removed. The array-gasket sandwich was separated from the chamber base by grabbing the slides from their ends. The microarray slide numeric barcode was kept facing up as the sandwich was transferred to slide-staining dish 1 containing Gene Expression Wash Buffer 1. The sandwich was opened from the barcode end whist submerged in Gene Expression Wash Buffer 1 and a forceps was used to release the slide form the gasket.

The slide was then placed into the slide rack in slide-staining dish 2 containing Gene Expression Wash Buffer 1 at room temperature. When all slides were placed in the slide rack, the rack was stirred on moderate speed for 5 minutes. The slide rack was transferred to slide-staining dish 3 containing Gene Expression Wash Buffer 2 at 37°C. It was stirred once again at moderate speed for 5 minutes. The slide rack was gently removed to minimise droplets and slides were transferred to a slide holder. The slides were scanned immediately to avoid effects on signal intensity by environmental oxidants.

2.6.4.9 Scanning and feature extraction

FIGURE 2-5

Image demonstrating the microarray mount for scanning⁴⁸⁵

The protocol from the Agilent microarray scanner user guide was used. The slide holders were placed into the SureScan cassette and miRNA settings were verified according to manufacturer’s recommendations (Figure 2-5). All original serum samples and additional endometriosis control samples were successfully processed. The attached Excel spreadsheets (see “Plate3_(1-53)_ExperimentalSummary.xlsx” and

“Plate1_(1-21)_ExperimentalSummary.xlsx”) outlines the sample hybridisation layout scheme for the respective sample sets (i.e. the slide number and array area used for each sample). The slides were successfully scanned (Agilent Scanner C, at 3 microns) and feature extracted (using Agilent Feature Extraction software, version 10.7.3).