Prism 7900

The concentrations of DNA samples were determined by the OD₂₆₀ and diluted to 20 ng/μl. Real-time PCR was performed using the Applied Biosystems Prism 7900HT sequence detection system with SDS 2.3 software. Each well contained 10 μl Sybr green (Applied Biosystems, Warrington, United Kingdom), 3.8 μl H₂O, 0.6 μl of 10 μM F primer, 0.6 μl of 10 μM R primer, and 5 μl sample (i.e., 20 ng DNA/well).

We selected four genes that were related to EMT and cancer stem cell markers based on the DEGs in our microarray data and a review of previous literature. Quantitative real‐time PCR was used to validate the expression levels of ALDH1A3, TM4SF1, PROM1, and CAV1. We designed primers with Primer Express Version 3.0 (Applied Biosystems) (Table 1). Real‐time qPCR analysis was performed on an Applied Biosystems Prism 7900 sequence detection system (PE Applied Biosystems, http://www.appliedbiosciences.com) with SYBR Green. Amplification conditions were the same for all primers: 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds, then 72°C for 30 seconds. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was the internal control standard. Experiments were independently performed in triplicate and qPCR cycle numbers were converted to gene amounts (ng) using an accepted formula.

From blood samples, DNA was isolated for genotyping using a QIAamp^® DNA blood kit according to the manufacturer’s instructions (Qiagen^®, Valencia, CA). The quality of isolated genomic DNA was assessed for each sample by agarose gel electrophoresis, while the quantity of DNA was determined by spectro-photometry (NanoDrop^®, Wilmington, USA). The studied NGFR Ser205Leu polymorphism (rs2072446) was genotyped by the TaqMan^® 5′-exonuclease assay using an Applied Biosystems^® (ABI^®, CA, USA) Prism 7900 instrument, as described previously19 (link).

Total RNA was extracted from the venous thrombi using the TRIzol reagent (Invitrogen). The cDNA was prepared from DNase-treated RNA (Applied Biosystems). Real-time PCR was performed with ABI PRISM 7900 using TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems) [17 (link)]. The following TaqMan Gene Expression Assays were used for each gene: assay ID Rn01538170_m1 for MMP-2, assay ID Rn00579162_m1 for MMP-9, assay ID Rn00565261_m1 for urokinase-type plasminogen activator (uPA), assay ID Rn01482578_m1 for tissue-type plasminogen activator (tPA), assay ID Rn01481341_m1 for plasminogen activator inhibitor-1 (PAI-1), assay ID Rn1462586_m1 for fibrinogen α chain, and assay ID Rn99999916_s1 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was used as an internal control.

To validate the array and to determine the angiogenic gene levels within the human endometrium, we performed complementary single-gene TaqMan quantitative RT-PCR analysis. In brief, a reaction mix was prepared containing TaqMan Supermix (5.5 mM MgCl₂, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP), ribosomal 18S primers/probe (Life Technologies, Carlsbad, CA, USA), and reaction-specific forward and reverse primers and probes (Universal Probe Library (Roche, Indianapolis, IN, USA) for CXCL2, CXCL8, CTGF, and VEGFC (Supplemental Table 3). A no-template control (with water instead of cDNA) was included on each plate. PCR was performed using an Applied Biosystems Prism 7900 instrument, and the results were analyzed in triplicate using Sequence Detector, version 2.3, and the 2ΔΔCt method [29 (link)]. Expression of target mRNA was normalized to RNA loading for each sample using 18S ribosomal RNA as an internal standard.