KUDZU EXTRACT AND PROCOLLAGEN PRODUCTION 477 MICROARRAY ANALYSIS—NORMAL HUMAN DERMAL FIBROBLAST CELL CULTURES Cultured normal human dermal fi broblasts (NHDF, Cascade Biologics) were grown in T-75 fl asks until confl uent using appropriate culture conditions. Upon reaching con- fl uence, the cells were treated with culture media supplemented with test materials at the concentrations specifi ed or with culture media alone, which acted as the untreated control. After applying the test materials to the cells, they were incubated for 24 hours at 37 ± 2°C and 5 ± 1% CO2. At the end of the incubation period, the culture media was re- moved by aspiration and the cells were washed once with cold phosphate-buffered saline using approximately 1 ml per well. After the washing step, a trypsin/EDTA solution was added to the wells to release the cells. After the cells were released, an appropriate vol- ume of trypsin-neutralizing solution was added to the wells. The treated cells and the untreated cells were pooled into separate 15-ml centrifuge tubes and pelleted by centri- fuging at 1000 rpm at 4 ± 2°C. After removing the supernatant, the pelleted cells were lysed by adding 300 μl of guanidinium thiocyanate lysis solution to each tube and then repeatedly drawing and releasing the solution into a pipet until the cell pellet dissolved. The cell lysate was then stored at −75°C until the RNA extraction process could be completed. RNA ISOLATION (AMBION RNAqueous KIT) To the thawed fi broblast lysate described above was added an equal volume of 64% etha- nol, and the culture tubes were mixed via vortex mixing. Seven hundred microliters of the alcoholic mixture was transferred to a glass-fi ber fi lter cartridge, the fi lter cartridge was loaded into a 1.5-ml collection tube, and the entire apparatus was centrifuged for 1 minute at 14,000 rpm. The fi ltration process was repeated until all of the mixture was fi ltered. The fi lter was then washed to remove any residual cellular debris from the RNA bound to the glass fi bers by subsequently applying 700 μl of wash solution followed by an additional two washes with 500 μl of wash solution, each time removing the wash solution by centrifuging the tube at 14,000 rpm for one minute. The fi ltrate was spun to remove any residual wash solvent. RNA bound to the glass fi bers within the cartridge was eluted by applying 30 μl of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pre- heated to 70–80°C) to the cartridge and centrifuging the cartridge in a new collection tube at 14,000 rpm for one minute. After the RNA had eluted, it was analyzed as de- scribed below. RNA CONCENTRATION ASSAY (Molecular Probes Ribogreen Assay) Ribogreen reagent was provided as a stock solution in DMSO. Prior to use, the reagent was diluted 2000-fold in TE buffer. The RNA assay requires 200 μl of diluted Ribogreen reagent per sample to be tested and 1 ml of the reagent for standards. Once prepared, the diluted reagent was stored in the dark. A series of RNA standards was prepared by diluting purifi ed ribosomal RNA derived from E. coli to the following concentrations: 2 μg/ml, 1 μg/ml, 200 ng/ml, 40 ng/ml, and 0 ng/ml (blank). Prior to assaying, the RNA samples, prepared as described above, were diluted 1000-fold in TE buffer. For the RNA assay, 100 μl of the diluted samples or standards

JOURNAL OF COSMETIC SCIENCE 478 was transferred to the wells of a black 96-well plate. The samples and standards were as- sayed in duplicate. After the samples/standards were added to the plate, 100 μl of the diluted Ribogreen assay reagent was added to the wells, and the plate was gently mixed and allowed to incubate for 5–10 minutes protected from light. After incubation, the plate was read by a fl uorometer using an excitation wavelength of 500 nm and an emis- sion wavelength of 525 nm. mRNA AMPLIFICATION (Ambion MessageAmp aRNA Kit) First-strand cDNA synthesis. Five micrograms of total RNA for each sample was placed into 600-μl PCR tubes, and the total volume of liquid in each tube was adjusted to 12 μl with DEPC·H2O. To each tube, 1 μl of T7 Oligo(dT) primer was added, and the tube was incubated at 70 ± 2°C for 10 minutes to denature the RNA. The sample was then placed onto ice to allow the primer to anneal to the poly A ends of the mRNA. After cooling, 2 μl of 10X fi rst-strand buffer, 1 μl of RNAse inhibitor, and 4 μl of dNTP mix was added to each tube and the tubes were placed at 42 ± 2°C. As soon as the tube was heated, 1 μl of reverse transcriptase was added and the tubes were returned to the 42 ± 22°C bath for two hours. After heating, the tubes were briefl y centrifuged to gather the fl uids at the bottom of the tube, and then they were cooled on ice. Second-strand synthesis and cDNA amplifi cation. For the synthesis of the second strand of cDNA, the following items were added to the tubes described above in the following order: 63 μl of DEPC·H2O, 10 μl of 10X second-strand buffer, 4 μl of dNPT mix, 2 μl of DNA polymerase, and 1 μl of RNAse H. Each tube was mixed and then incubated at 16 ± 2°C for two hours. Toward the end of the two-hour incubation, a suffi cient quantity of DEPC·H2O was added, the tube was warmed to 50 ± 2°C, and a cDNA purifi cation fi lter cartridge was equilibrated with 50 μl of cDNA binding buffer (one cartridge per sample) for at least fi ve minutes. After the samples had fi nished incubating, 250 μl of cDNA binding buffer was added to each tube and the tubes were thoroughly mixed. The contents of the each PCR tube were transferred to the cDNA purifi cation fi lter cartridge. The cartridge was then placed in a collection tube and centrifuged at 10,000 rpm for one minute. The fl ow-through was discarded and 650 μl of cDNA wash solution was added to the cartridge. The cartridge was centrifuged again and the fl ow-through was discarded. The contents were centrifuged one more time to ensure that the wash buffer was completely emptied from the fi lter. The cDNA was eluted by applying 10 μl of preheated DEPC·H2O to the fi lter, and the contents were centrifuged in a new collection tube at 10,000 rpm for one minute. The elution was repeated one additional time to give a total volume of 16–18 μl of cDNA. In vitro transcription to synthesize aRNA and aRNA purifi cation. The in vitro transcription began by adding the following to the cDNA solution: 4 μl of 10X reaction buffer and 4 μl of T7 enzyme mix. The tube was mixed and then incubated at 37 ± 2°C for 6–14 hours. Towards the end of the incubation period, a suffi cient volume of elution solution was warmed to 50°–60°C and an aRNA fi lter cartridge was equilibrated with 100 μl of aRNA binding buffer for at least fi ve minutes. At the end of the incubation period, 350 μl of aRNA binding buffer was added to the sample tubes and thoroughly mixed. An additional 250 μl of absolute ethanol was added to each tube. The mixture was trans- ferred to an aRNA fi lter cartridge, and the cartridge was then inserted into a collection tube and centrifuged at 10,000 rpm for one minute. The fl ow-through was discarded and 650 μl of aRNA wash buffer was added to the cartridge, followed by further centrifugation