Tumor was harvested from animals at day eight of the treatment. RNA was extracted using RNeasy kit.
Biomaterial provider
own
Treatment protocol
not treated
Growth protocol
mice were grown in CINJ facility, standard condition
Extracted molecule
total RNA
Extraction protocol
RNeasy kit from Qiagen
Label
biotin
Label protocol
Synthesis of Biotin-Labeled cRNA Thaw components of the ENZO BioArray RNA Transcript Labeling Kit and purified cDNA mixture from the previous day. In a new, sterile, 1.5mL RNase-free microfuge tube, add the following components:
Gently mix and spin down. Incubate in a 37°C water bath for 4 hours.
Cleanup of IVT Reaction
Take out 1ul of the reaction mix for gel electrophoresis inspection. Using the GeneChip Sample Cleanup Module, add 60µl of RNase-free water to the reaction. Mix for 3 seconds. Add 350µl IVT cRNA Binding Buffer and mix for 3 seconds. Add 250µl of 100% ethanol and mix well (do not centrifuge). Add sample (~700ul total) to the IVT cRNA Cleanup Spin Column sitting in a 2mL Collection Tube. Centrifuge for 15 seconds at 10,000rpm. Discard flow-through and Collection Tube. Transfer spin column to a new 2mL Collection Tube. Add 500µl IVT cRNA Wash Buffer to spin column. Centrifuge for 15 seconds at 10,000rpm. Discard flow-through. Add 500µl of 80% ethanol onto spin column. Centrifuge for 15 seconds at 10,000rpm. Discard flow-through. Open the cap of the spin column and centrifuge for 5 minutes at max. speed. Discard flow-through and Collection Tube. Transfer spin column to a new 1.5mL Collection Tube and pipet 11µl of RNase-free water directly onto the spin column membrane. Centrifuge for 1 minute at max. speed to elute. Add another 10µl of RNase-free water directly onto spin column membrane and centrifuge for 1 minute at max. speed to elute.
Use spectrophotometric analysis to determine cRNA yield and purity using a 1:100 dilution of purified cRNA (A260/A280 ratio should be close to 2.0 for pure RNA).
Calculate the adjusted cRNA yield using the following:
adjusted cRNA yield = RNAm (total RNAi)(y)
RNAm = amount of cRNA measured after IVT (µg) total RNAi = starting amount of total RNA (µg) y = fraction of cDNA reaction used in IVT
For fragmentation, adjusted cRNA concentration must be at least 0.6µg/µl. The final concentration of RNA in the fragmentation mix can range from 0.5µg/µl to 2µg/µl.
For standard arrays, 15µg of fragmented cRNA is required. Fragmentation In a thermal cycler compatible microfuge tube, combine 4µl 5X Fragmentation Buffer (supplied in GeneChip Sample Cleanup Module), 15µg cRNA (variable) and RNase-free water for a final volume of 20µl. Incubate in a thermal cycler at 94°C for 35 minutes. Place mixture on ice. Take out 1ul of fragmented cRNA for gel electrophoresis inspection.
Run crude cRNA and fragmented cRNA samples, along with a 1Kb ladder on a 1% agarose gel for visual inspection.
Hybridization protocol
Hybridization -Thaw components of the Eukaryotic Hybridization Control Kit. Heat 20X Eukaryotic Hybridization Control to 65°C for 5 minutes to denature cRNA controls. Equilibrate desired probe array to room temperature. In a new, sterile RNase-free 1.5mL microfuge tube, add the following components:
-20µl fragmented cRNA -5µl Control Oligonucleotide B2 (3nM) -15µl 20X Eukaryotic Hybridization Controls -3µl Herring Sperm DNA (10mg/mL) -3µl Acetylated BSA (50mg/mL) -150µl 2X Hybridization Buffer -104µl RNase-free water
Total hybridization volume is 300µl
Heat hybridization cocktail to 95°C for 5 minutes, followed by heating at 45°C for 5 minutes. In a new, sterile RNase-free 1.5mL microfuge tube, combine a volume of 2X Hybridization Buffer with an equal volume of RNase-free water (make 1X Hybridization Buffer). Wet the probe array by filling it with 250µl of 1X Hybridization Buffer and incubate in Hybridization Oven 640 set at 45°C for 10 minutes with rotation setting at 60rpm. Centrifuge hybridization cocktail for 5 minutes at max. speed to spin down any insoluble material. Remove 1X Hybridization Buffer from probe array and fill with 250µl of hybridization cocktail, taking care not to pipette any insoluble material. Place probe array in Hybridization Oven 640, heated to 45°C and with rotation setting at 60rpm for 16 hours.
Scan protocol
Washing, Staining, and Scanning
Stain
For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:
600.0µl of 2X MES Stain Buffer 48.0µl Acetylated BSA (50mg/mL) 12.0µl Streptavidin Phycoerythrin (SAPE) (1mg/mL) 540.0µl water, Molecular Biology Grade
Total volume is 1200µl.
Divide into two aliquots of 600µl each to be used for stains 1 and 3.
Antibody For each array, in a new, sterile RNase-free 1.5mL microfuge tube, combine the following:
After 16 hours of hybridization, remove 250µl of sample (in hybridization cocktail) and put back into eppendorf, containing fragmented cRNA sample. Fill the array with 250µl of Non-Stringent Wash Buffer (Wash A)
Now ready for Fluidics Station, followed by scanning.
Description
Protocol taken from the web site of CINJ Core Expression Array Facility where the samples were processed.
Data processing
scaned fluorescence intensity values, no normalization
