2.4. Synthesis of cRNA, Microarray Hybridization and Scanning
Total RNA samples from mesocarp were individually treated and labeled with a one-color (Cy3) dye according to the Low Input Quick Amp Labeling protocol (version 6.0; December 2009) provided by Agilent. A total of 100 ng of total RNA was used to synthesize cRNA labeled with Cy3 dye. Total RNA of 100 ng in 1.5 µL was mixed with 2 µL of Agilent One-Color Spike Mix. T7 promoter primer was added into the mixture and the reaction made up with Nuclease-free water to 5.3 µL. The reaction mixture was incubated at 65 °C for 10 min using a thermal cycler (BIO-RAD, C-1000, Hercules, CA, USA). After incubation, the reaction mixture was incubated on ice for 5 min. To reverse transcript the mRNAs to cDNA, a total of 4.7 µL cDNA Master mix was added to the previous reaction mixture. This consisted of 2 µL of 5X first strand buffer, 1 µL of 0.1 M DTT, 0.5 µL of 10mM dNTP mix and 1.2 µL of AffinityScript RNase Block mix. The reaction mixture was mixed thoroughly by pipetting up and down. After briefly centrifuging, the reaction mixture was incubated at 40 °C for 2 h, followed by 70 °C for 15 min using a thermal cycler (C1000, BIO-RAD). After the cDNA synthesis process, the reaction mixture was kept on ice for 5 min, before 6 µL of transcription mixture was added giving a total volume of 16 µL. This step was performed to transcribe cDNA to cRNA and incorporated the Cyanine 3-CTP dye to cRNA during the transcription process. The transcription mix consist of 3.2 µL of 5X transcription buffer, 0.6 µL of 0.1 M DTT, 1 µL of NTP mix, 0.21 µL of T7 RNA Polymerase Blend, 0.24 µL of Cyanine 3-CTP, made up with nuclease-free water to a volume of 6 µL. The reaction was mixed and incubated at 40 °C for 2 h using a thermal cycler. Labeled cRNAs were then purified using Qiagen’s RNeasy mini kit (Hilden, Germany) as recommended by Agilent. The quality and quantity were assessed using the Spectrophotometer ND-1000 (Thermo Scientific). A total of 1.65 µg of purified labeled cRNA was used for hybridization. The purified cRNA was mixed with 25 µL of 10X blocking Agent, 5 µL of 25X Fragmentation buffer and made up to a volume of 120 µL with nuclease-free water. The reaction was mixed and incubated at 60 °C for 30 min to fragment the cRNA and cooled on ice immediately after fragmentation. A total of 125 µL 2X GE Hybridization Buffer HI-RPM was mixed with the fragmented cRNA and hybridized onto the arrays at 65 °C for 16 h in a rotating hybridization oven. After hybridization, 2 steps of washing were performed with wash buffer 1 and 2 for 1 min each. The array was then air-dried for a few seconds before proceeding with image scanning using the Agilent microarray slide scanner (SG11350602). The slides were scanned using the green dye channel with scanning resolution of 5 µm at 20 bit of dynamic range.