array scanning and feature extraction protocol
P-AFFY-6 Affymetrix CEL analysis
Title: Affymetrix CHP Analysis (ExpressionCall). Description:
nucleic acid hybridization to array protocol
The cRNA was then added to a hybridization cocktail containing salts, blocking agents and hybridization controls. The hybridization controls are composed of a mixture of biotin-labeled cRNA transcripts of bioB, bioC, bioD, and cre, prepared in staggered concentrations (1.5 pM, 5 pM, 25 pM, and 100 pM final concentrations for bioB, bioC, bioD, and cre, respectively) (GeneChip Expression Analysis Technical Manual). The cocktail was injected into the Genechip hybridization Oven 640 (Affymetrix, Santa Clara, CA, U.S.A.) and hybridized to the GeneChip at 45 degrees Celsius for 16 hours. (Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 1, Mass unit = Micro gram, Tiny time unit = seconds, Volume unit = Nano litre, temperature = 45)
In each experiment, RNA was extracted from tomato leaf tissue sample subjected to the four treatments. Total RNA was pooled for each treatment (3 – 4 experimental replicates, i.e., plants) within a biological replication or experiment.
nucleic acid labeling protocol
The RNA labeling process was performed using the One-cycle target labeling/3’ IVT assay. Briefly, total RNA (1 ug to 15 ug) was reverse transcribed using reverse transcriptase and an T7-Oligo(dT) Promoter Primer to produce double stranded cDNA. The cDNA then served as a template in an in vitro transcription (IVT) reaction in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling (GeneChip Expression Analysis Technical Manual). The biotinylated cRNA targets are then cleaned up, fragmented using heat and Mg+2, and hybridized to the GeneChip. cRNA was then added to a hybridization cocktail containing salts, blocking agents and hybridization controls. (Parameters: Amplification = none, Mass unit = Micro gram)
Tomato plants were grown in 15.2-cm pots filled with Metro mix potting soil and each pot was housed in a thrips-proof-screened (No Thrips Insect Screen, BioQuip Products, Rancho Dominguez, CA) cage in a greenhouse. Temperatures ranged from 23-25C and the photoperiod was 16:8 h (light:dark). Plants were fertilized once a week with Miracle Gro-Water Soluble All Purpose Plant Food (24-8-16) NPK. (Parameters: time unit = seconds, min temperature = 23, max temperature = 25, temperature unit = C)
nucleic acid extraction protocol
Total RNA was isolated from frozen leaflet samples (100 – 200 mg of tissue) using the Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A) following manufacturers protocol. RNA concentrations were determined by NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and quality assessed with the 2100 Bioanalyzer using Nanochip technology (Agilent Technologies, Inc., Palo Alto, CA, USA). (Parameters: Extracted product = total_RNA, Amplification = none)
Five-week old tomato plants were subjected to four treatments: 1) mock-inoculated, 2) virus-infected, 3) spider mite infestation, and 4) virus + spider mite infestation. Plant tissue was harvested from each treatment after 7-days, frozen in liquid nitrogen and stored in -80 degree celsius until use.