normal tissue from mastectomy specimen from postmenopausal patient with invasive lobular carcinoma (ILC)
Biomaterial provider
Institute of Pathology, Palacky University, Czech Republic
Extracted molecule
total RNA
Extraction protocol
At least 1000 normal ductal cells were microdissected from cryosections using the VeritasTM Laser Capture Microdissection System (Arcturus Bioscience, Inc., USA) according to standard protocols. Caps with captured cells were directly placed in 100 ul lysis buffer (Qiagen, Hilden, Germany). Total cellular RNA was isolated (RNeasy® Micro Kit, Qiagen) according to manufacturer´s recommendations and subsequently quantified on a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).
Label
biotin-14-CTP (Invitrogen, CarlsBad, CA, USA) and biotin-16-UTP (Roche)
Label protocol
Fifty nanograms of total RNA were reverse transcribed and amplified by Microarray Target Amplification Kit (Roche diagnostics, Basel, Switzerland). In brief, total RNA (50 ng) was converted into cDNA using a modified oligo (dT) primer (TAS-T7 Oligo (dT)24). A unique Target Amplification Sequence (TAS) with no homology to any known sequences in public databases generated the 3´ anchor on the cDNA for subsequent PCR amplification. In order to include a 5´ anchor sequence on the cDNA, the TAS-(dN)10 primer was used for the initiation of the second strand cDNA synthesis. After purification using the Microarray Target Purification Kit (Roche), PCR was performed using the TAS primer and Expand PCR Enzyme Mix which is optimized for long (>1kb) and unbiased PCR products. In order to ensure that messages were not amplified to saturation, the optimal number of PCR cycles was estimated by preliminary PCR and agarose electrophoresis of PCR products from cycles 21, 24, 27, 30 and 33. After purification with the Microarray Target Purification Kit (Roche), the PCR products were labeled with biotin-14-CTP (Invitrogen, CarlsBad, CA, USA) and biotin-16-UTP (Roche) by in vitro transcription using Microarray RNA Target Synthesis Kit T7 (Roche). The labelled cRNA was purified using the Microarray Target Purification Kit (Roche), quantified by spectrophotometer and checked by agarose electrophoresis. The entire amplification and labelling process was monitored by GeneChip® Eukaryotic Poly-A RNA Control Kit (Affymetrix, Santa Clara, CA, USA) with exogenous positive controls which were spiked into the total RNA before cDNA synthesis and finally detected on Human Genome U133 Plus 2.0 Arrays (Affymetrix).
Hybridization protocol
The biotinylated cRNA preparation was fragmented, assessed by gel electrophoresis, and placed in hybridization cocktail containing biotinylated hybridization controls (GeneChipTM Eukaryotic Hybridization Control Kit, Affymetrix). Sample was first hybridized to Test3 Arrays for 16 hours, washed, stained using antibody-mediated signal amplification and scanned. After passing this quality control stage, the sample was hybridized onto the large Human Genome U133 Plus 2.0 Array.
Scan protocol
see associated EXP file
Description
Amplified total RNA from microdissected normal lobular cells from mastectomy specimen from postmenopausal patient with invasive lobular carcinoma (ILC)
Data processing
GCOS with the default settings exept that the target signal was set to 100
