|Public on Jan 29, 2014
|subject id: 5
tissue: vaginal tissue
tissue location (a-p): Anterior
tissue location (p-d): Proximal
tissue location (l-r): Right
|Samples were thawed on ice and mixed by repeat pipet disruption with 70% ethanol according to the procedure described in the technical protocol of the RNeasy Mini Kit (QIAGEN), which was used for subsequent purification according to the manufacturer instructions. This included an on-column DNase digestion with the RNase-free DNase kit (QIAGEN). The total RNAs were eluted once with 50 µL of RNase-free water.
|Cyanine-3 (Cy3) labeled cRNA was prepared from 25ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|600ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 arrays (G4851B, Design ID 039494) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then immediately put the slides with Agilent barcode facing up in a slide holder with an ozone-barrier slide cover on top of the array.
|Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60K array(Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%). Raw data were extracted using the Agilent Feature Extraction software, version 10.7.3.1., protocol GE1_107_Sep09.
|vaginal tissue sample
|Microarray expression values were calculated using the gProcessed Signal, which was normalized via log2 transformation and quantile normalization in Partek® Genomics Suite (v.6.6, Partek Inc, St. Louis, MO). Control probe expression levels were removed during normalization. Expression changes due to batch effects between arrays have been removed.
|Aug 14, 2013
|Last update date
|Jan 29, 2014
|Charles David Warden
|City of Hope
|Department of Molecular and Cellular Biology
|1500 East Duarte Rd
|Global Expression of Molecular Transporters in the Human Vaginal Tract