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Blood, Vol. 111, Issue 6, 3200-3210, March 15, 2008
Pathway analysis of primary central nervous system lymphoma
Blood Tun et al.
111: 3200
Supplemental materials for: Tun et al
Supplemental materials and methods Detailed protocol for quantitative Real-Time PCR (qPCR)
A portion of the total RNA extracted from the lymphoma tissue was used for validation by qPCR. Validation of microarray results was performed for ten genes on aRNA transcribed from double-stranded (ds) cDNA from the second stage of T7 amplification. The aRNA produced in this reaction was converted to cDNA using reverse transcriptase and random hexamer priming. First-strand cDNA synthesis of one microgram of amplified transcripts (aRNA, see in vitro transcription method above) was carried out according to kit instructions (Invitrogen, Calsbad, CA, random primer method) with slight modifications to the protocol. We found that cDNA synthesis of aRNA improved when the incubation temperature was increased from 50 deg C to 55 deg C. Thirteen (13) µL of a qRT-PCR reagent master mix (SuperArray, Fredrick, MD) 6.25 µL SYBR Green PCR Master Mix, 0.5 µL appropriate primer set, and 5.25 µL molecular biology-grade water (Eppendorf, Westbury, NY) per reaction were combined in a nucleic acid-, nuclease-free microfuge tube (Ambion, Austin, TX), mixed in an sterile round bottomed polystyrene microtiter plate well (Greiner Bio-One, Durham, NC) containing 1 µL cDNA and subsequently transferred to a 384-well Clear Optical Reaction Plate (ABI Prism, Foster City, CA). The reaction was performed according to the company protocol (SuperArray) for all primer sets with a slight modification: 95 deg C, 15 min; then 95 deg C, 30 s, 55 deg C, 30 s, 72 deg C, 45 s for 50 cycles. Real-time PCR amplicon detection was performed on an ABI Prism Sequence Detection System, model 7900HT, and the data captured with the ABI Prism SDS v2.1 software. Ten primer sets (SuperArray) targeting the following gene transcripts of interest: ATP5J (NM_001685.4), BCL-6 (NM_001706.2), CD10 (NM_000902.3), CD44 (NM_000610.3), CHI3L1 (NM_001276.1), COX6B1 (NM_001863.3), IRF4 (NM_002460.1), SPP1 (NM_000582.2), TFPI2 (NM_006528.2) and GAPDH (NM_002046.3). Log serial dilutions of pooled cDNA samples were assayed by PCR to determine amplification efficiencies for the genes of interest and the “housekeeping” gene GAPDH. Threshold cycle (Ct) values were extrapolated by the Prism software to quantify relative gene expression values. Detailed protocol for immunohistochemistry (IHC) procedures
Sections (5 µm) were cut from paraffin-embedded tissue blocks and mounted on silanized slides and dried at 60 deg C for 1 hour Paraffin was removed with three (3) washes for 5 min with xylene. The sections equilibrated twice with absolute ethanol, then rehydrated with 95% ethanol and tap water. Sections were equilibrated with phosphate-buffered saline (PBS) containing Tween 20 for 5 min, then placed into hot antigen retrieval solution (steam retrieval) at pH 6.0 (DAKO Cytomation, Carpenteria, CA) for 20 minutes and cooled to room temperature over another 20 minutes. Endogenous peroxidases were blocked by treatment with 3% hydrogen peroxide for 5 min, followed by rinsing in PBS w/Tween (DAKO) 20 for 5 minutes. A DAKOCytomation Immunostainer Plus (DAKO) unit was used for subsequent steps. At immersions in Non-Serum Blocking solution (DAKO) for 5 minutes, sections were treated 2 hr with either anti-osteopontin antibody (R&D Systems, Inc. Minneapolis, MN; 1:10 dilution) or anti-CHI3L1 antibody (Quidel Corporation, San Diego, CA; 1:140 dilution) at RT. Sections were transferred either to Envision Dual-labeled Polymer Plus Labeling System (DAKO; for osteopontin) or rabbit-labeled Polymer Plust Labeling System (DAKO, for CHI3L1) for 30 min. Sections were reacted with diaminobenzidine/hydrogen peroxide for 10 min, counterstained with Gill 1 Hematoxylin (Richard Allen Scientific/Thermo Fisher) for 1 min, and coverslipped using #1 coverslips and Cytoseal mounting media (Thermo/Fisher). Digital microscopy was performed with a SPOT RT Color Camera (Diagnostics Instruments) mounted on a Leica DMLB microscope; images were processed using the SPOT Advanced program and Adobe Photoshop CS.
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