Plants were grown in hydroponic conditions in growth chamber at 28oC /16 hours light and 20oC/8 hours dark cycle.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Qiagen plant RNeasy extraction kit according to manufacturer's protocol.
Label
biotin
Label protocol
The labeling of the RNA was done as per the manufacturer's instructions (Affymetrix, Santa Clara, CA). Briefly, two microgram of total RNA was reverse transcribed using an oligo dT-T7 primer under standard conditions. The resulting first strand cDNA was incubated with RNAse H, E. coli DNA ligase, and E. coli DNA polymerase, dNTPs and buffer to produce second-stranded cDNA. The cDNA was purified using spin columns following the manufacturer’s instructions (Qiagen). The purified cDNA was then used in an in vitro transcription assay to produce the target biotin labeled cRNA for hybridization using the 3’ IVT kit from Affymetrix following the manufacturer’s instructions. The cRNA was purified using a spin column following the kit’s instructions (Qiagen), and fragmented prior to hybridization onto the GeneChips.
Hybridization protocol
Hybridization of the GeneChips was carried out following the manufacturer’s protocol. Briefly, the fragmented biotin labeled cRNA was mixed with 0.05nM control oligonucleotide B2, 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 0.1 mg/mL herring sperm DNA, 0.5 mg/mL BSA, 2X hybridization buffer (1X 100mM MES/1M NaCl/20mM EDTA/0.01% Tween-20) and 10% DMSO. The hybridization cocktail was incubated at 99oC for 5 minutes to denature the herring sperm DNA and any secondary structure on the cRNA. The cocktail was then equilibrated to 45oC for 5 minutes prior to addition to the GeneChip. Hybridization was carried out in the Affymetrix Hybridization Oven 640 at 45oC with rotation at 60rpm for 16 hours. Following the hybridization step the GeneChips were washed and stained in the Affymetrix Fluidics Station 450 following Fluidics Protocol Mini_euk2v3 as recommended by the manufacturer for this Chip type. The GeneChips were first washed in a non-stringent wash buffer of 6X SSPE/0.01% Tween-20 followed by stringent wash with 100mM MES/0.1M NaCl/0.01% Tween-20. The first staining of the GeneChips was done in 2X stain buffer (1X 100mM MES/1M NaCl/0.05% Tween-20), 2mg/mL BSA, and 0.01 mg/mL Streptavidin Phycoerythrin (SAPE). The GeneChips were washed in non-stringent wash buffer. A second stain comprised of 2X staining buffer, 2mg/mL BSA, 0.1mg/mL Goat IgG, and 0.003mg/mL of biotinylated antibody was conducted immediately followed by a third stain comprised of the SAPE solution (see above). Finally, the GeneChips were washed in non-stringent wash buffer prior to scanning.
Scan protocol
Scanning of the GeneChips was done using the Affymetrix 3000 7G scanner as described by the manufacturer. The Fluidics station and Scanner were controlled using Affymetrix GeneChip Command Console (AGCC).
