Adhesive patches were macrodissected to separate lesional tissue from surrounding normal tissue. Total RNA was isolated from the recovered lesional tissue using a modified PicoPure procedure (Life Technologies, Foster City, CA) and reverse transcribed to complementary DNA using SuperScript VILO complementary DNA synthesis kits (Thermo Fisher Scientific, Pittsburgh, PA). The resulting complementary DNA was subsequently used for target gene expression analysis with quantitative real-time (qRT)-polymerase chain reaction (PCR) on an ABI7900 PCR system (Life Technologies). Each qRT-PCR reaction used 3 pg of total RNA, in duplicate, in 20-μL volume on 384-well PCR reaction plates using predesigned gene-specific TaqMan probe chemistries (Life Technologies). An averaged cycle threshold value of the duplicate measurements was used in the analysis. Gene expression was considered detected if the quantitative polymerase chain reaction yielded an amplification curve and a measurable cycle threshold value, or not detected if the reaction yielded an undetermined cycle threshold value (amplification curve never above detection threshold). In addition to the 2 target genes, human β-actin was used as an internal control.
Taqman probe chemistries
Manufactured by Thermo Fisher Scientific
TaqMan probe chemistries are a set of fluorescent-based detection systems used in real-time PCR (qPCR) assays. They employ a DNA probe labeled with a reporter dye and a quencher dye. During the PCR amplification process, the probe specifically binds to the target DNA sequence, and the reporter dye is cleaved, emitting a fluorescent signal that is proportional to the amount of amplified product.
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2 protocols using taqman probe chemistries
1
Quantitative Real-Time PCR Tissue Analysis
2
Quantitative Real-Time PCR Tissue Analysis
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Adhesive patches were macrodissected to separate lesional tissue from surrounding normal tissue. Total RNA was isolated from the recovered lesional tissue using a modified PicoPure procedure (Life Technologies, Foster City, CA) and reverse transcribed to complementary DNA using SuperScript VILO complementary DNA synthesis kits (Thermo Fisher Scientific, Pittsburgh, PA). The resulting complementary DNA was subsequently used for target gene expression analysis with quantitative real-time (qRT)-polymerase chain reaction (PCR) on an ABI7900 PCR system (Life Technologies). Each qRT-PCR reaction used 3 pg of total RNA, in duplicate, in 20-μL volume on 384-well PCR reaction plates using predesigned gene-specific TaqMan probe chemistries (Life Technologies). An averaged cycle threshold value of the duplicate measurements was used in the analysis. Gene expression was considered detected if the quantitative polymerase chain reaction yielded an amplification curve and a measurable cycle threshold value, or not detected if the reaction yielded an undetermined cycle threshold value (amplification curve never above detection threshold). In addition to the 2 target genes, human β-actin was used as an internal control.
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