Computational protocol: Evaluation of the Prognostic Value of RANK, OPG, and RANKL mRNA Expression in Early Breast Cancer Patients Treated with Anthracycline-Based Adjuvant Chemotherapy

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Protocol publication

[…] Prior to RNA isolation, macrodissection of tumor areas was performed in most (69%) of the FFPE sections (all sections with <50% tumor cell content). More than one FFPE section (2-8 sections, 10 μm thick) was used for RNA extraction when the tumor surface of a given sample was less than 0.25 cm2. From each FFPE section or macrodissected tissue fragments, RNA was extracted using a standardized fully automated isolation method for total RNA from FFPE tissue based on germanium-coated magnetic beads (XTRAKT kit; STRATIFYER Molecular Pathology GmbH, Cologne, Germany) in combination with a liquid handling robot (XTRAKT XL; STRATIFYER Molecular Pathology GmbH), as previously described in detail , , , , . The method involves extraction-integrated deparaffinization and DNase I digestion steps. The quality and quantity of RNA were checked by measuring CALM2 expression as a surrogate for amplifiable mRNA by qRT-PCR. CALM2 was used as endogenous reference because it had previously been identified as being highly and stably expressed among breast cancer tissue samples. Of the 975 FFPE tumor tissue samples collected, 819 (84.0%) had enough material left for RNA isolation needed for the present study.qRT-PCR primers and labeled hydrolysis probes were selected using Primer Express Software, Versions 2.2 and 3 (Applied Biosystems/Life Technologies, Karlsruhe, Germany), according to the manufacturer's instructions, and were controlled for single nucleotide polymorphisms. All primers, probes, and amplicons were checked for their specificity against nucleotide databases at NCBI using Basic Local Alignment Search Tool. Primers and probes were purchased from Eurogentec S.A. (Seraing, Belgium). For each primer/probe set, the amplification efficiency was tested, aiming to reach comparable efficiency of >90% (efficiency range from 97.7 to 99.7%). Primers and hydrolysis probes were diluted to 100 μM using a stock solution with nuclease-free water (Life Technologies GmbH, Darmstadt, Germany) , , . qRT-PCR was applied for the relative quantification of RANK, OPG, and RANKL. The Primer/Probe (YakimaYellow/FAM-labeled) sets used for amplification of the target and reference genes are shown in .For PCR, 0.5 μM of each primer and 0.25 μM of each probe were used. All qRT-PCRs were performed in triplicates using the SuperScript III Platinum One-Step qRT-PCR kit (Invitrogen/Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions. Experiments were performed on a Stratagene Mx3005p (Agilent Technologies, Waldbronn, Germany) with 30 minutes at 50°C and 2 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C. The lengths of the amplicons detected by the RANK, OPG, RANKL, and CALM2 assays were 106 bp, 83 bp, 72 bp and 72 bp, respectively, with PCR efficiencies [E = 1(10 − slope)] of 93.5%, 101.6%, 101.7%, and 99.7%, respectively. Samples were considered eligible for further investigation (N = 814, ) when the cycle threshold (CT) values of the housekeeping gene were ≤33.5 (triplicate mean values). Relative expression levels (relative quantification) of the target transcripts were calculated as 40 − DCT values (DCT = mean CT target gene − mean CT housekeeping gene) to yield positively correlated numbers and to facilitate comparisons , , . OPG and RANKL results were available for all 814 eligible samples, whereas RANK results were available for 784 patients because of inadequate amount of RNA extract in 30 samples, in which only OPG and RANKL were evaluated. A commercially available human reference RNA (Stratagene qPCR Human Reference Total RNA; Agilent Technologies) was used as positive control. No-template controls were assessed in parallel to exclude contamination. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Application qPCR
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms
Chemicals Estrogens, Progesterone, Anthracyclines