kindly provided by Dr. P. Boukamp, DKFZ, Heidelberg, Germany through Dr. T.C., Lee, SINICA, Taipei, Taiwan
Treatment protocol
Cells exposed to 5J/m2 UVB follow by15G ELF-EMF for 4hours
Growth protocol
Cells were maintained at 37 oC and 5 % CO2 /95% air in DMEM (Gibico) supplemented with 2mM L-glutamine, 100 U/ml penicillin/ 100μg/ml streptomycin(Gibico), and 10% fetal bovine serum(Hyclone). Cells were routinely split 1: 6 every 3 days and have a plating efficiency of approximately 10%. For microarray experiments, 2×106 cells were seed 48 hours before treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracte by lying directly in the 100mm petri dish with 1.5 ml TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction and then purified by RNeasy kit (Qiagen, Inc., Valencia, CA).
Label
dUTP-Cy5
Label protocol
90μg total RNA and 7.34ng spike RNA was annealed to oligo(dT) and reversely transcribed by Superscript II (Invitrogen, Carlsbad, CA) to single strand cDNA labeled with Cy5-labeled dUTP (Amersham Pharmacia Biotech, Piscataway, NJ). Reactions were terminated using 0.1 N NaOH and then neutralized by 0.1 N HCl. Labeled cDNA was purified by microcon YM-30 column (Qiagen, Inc., Valencia, CA) and blocked by adding 20μg of Cot1 human DNA (Invitrogen, Carlsbad, CA), 20μg ployA RNA (Invitrogen, Carlsbad, CA) and 20μg tRNA (Invitrogen, Carlsbad, CA).
kindly provided by Dr. P. Boukamp, DKFZ, Heidelberg, Germany through Dr. T.C., Lee, SINICA, Taipei, Taiwan
Treatment protocol
NoCells exposed to 5J/m2 UVB
Growth protocol
Cells were maintained at 37 oC and 5 % CO2 /95% air in DMEM (Gibico) supplemented with 2mM L-glutamine, 100 U/ml penicillin/ 100μg/ml streptomycin(Gibico), and 10% fetal bovine serum(Hyclone). Cells were routinely split 1: 6 every 3 days and have a plating efficiency of approximately 10%. For microarray experiments, 2×106 cells were seed 48 hours before treatment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracte by lying directly in the 100mm petri dish with 1.5 ml TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instruction and then purified by RNeasy kit (Qiagen, Inc., Valencia, CA).
Label
dUTP-Cy3
Label protocol
90μg total RNA and 7.34ng spike RNA was annealed to oligo(dT) and reversely transcribed by Superscript II (Invitrogen, Carlsbad, CA) to single strand cDNA labeled with Cy3-labeled dUTP (Amersham Pharmacia Biotech, Piscataway, NJ). Reactions were terminated using 0.1 N NaOH and then neutralized by 0.1 N HCl. Labeled cDNA was purified by microcon YM-30 column (Qiagen, Inc., Valencia, CA) and blocked by adding 20μg of Cot1 human DNA (Invitrogen, Carlsbad, CA), 20μg ployA RNA (Invitrogen, Carlsbad, CA) and 20μg tRNA (Invitrogen, Carlsbad, CA).
Hybridization protocol
Microarray was first blocked by 0.1 mg/ml BSA, 3xSSC, and 0.1% SDS mixed solution for 1 hours at 50 oC. Then dipped in ddH2O for four times. Labeled cDNA was pipetted onto the center of the microarray and a coverslip (large enough to cover the entire array surface) was placed over the probe. The microarray was transferred in a hybridization chamber and four 16μl drops of 3x SSC were pipetted around the slide to provide humidity in the hybridization chamber (Takara, Japan). Then, the hybridization chamber was submerged in a 65℃ water bath for 16 to 20 hours. After hybridization was complete, the microarray was washed in 2x SSC and 0.1% SDS at 42°C for 5 min, followed by additional 10 min wash steps in 0.1x SSC and 0.1% SDS, 1mim in 0.1x SSC for four times, and 10s in 0.01 x SSC, each at room temperature.
