Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction

Abstract

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure.We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)a, ERb, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene.A good correlation was observed for all target and reference genes in both centers using their own protocols and kits ðP < 0:0001Þ. The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERa and the PR transcripts ðP < 0:001Þ. A weak inverse correlation was observed in both centers between ERa and ERb levels, but only when TBP was the reference gene. No other correlation was observed with other parameters.Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of interlaboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

“…The necessity for ensuring quality-assurance measures for qPCR and RT-qPCR assays is well recognized (25,44,(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(86). The main difference between qPCR and conventional (legacy) PCR assays is the expectation of the former's potential to quantify target nucleic acids accurately.…”

A New Model of Patient Tumor-Derived Breast Cancer Xenografts for Preclinical Assays

“…Our experience shows that the interscapular fat pad provides higher tumor take than orthotopic sites: using double implantation (mammary fat pad and interscapular area) in the same mouse, we observed differences of tumor engraftment in favor for the interscapular fat pad. 7 Implantation in the renal capsule might be susceptible to promote growth as a result of the rich vascularity (12) and could lead to an increase in the take especially of low-grade ER-positive tumors, which are very difficult to maintain in serial passages. (c) The hormone status of breast tumors is a critical parameter: ER-negative tumors show a significant superiority of tumor take when compared with ER-positive tumors.…”

“…Moreover, a high correlation between drug response of tumor xenografts was found with the corresponding original tumors, with a positive prediction of 90%, which is much higher that the predictive value found with in vitro models, as reported by Fiebig's group (2), and observed by us. 7 However, few models of breast cancer xenografts are currently available; hence, the well-known diversity of these cancers is not represented in existing preclinical models. For some human carcinomas, such as non -small cell lung cancers or colorectal cancers, the establishment of human tumor xenografts in nude mice has been achievable with a success rate of more than 50%.…”

“…For some human carcinomas, such as non -small cell lung cancers or colorectal cancers, the establishment of human tumor xenografts in nude mice has been achievable with a success rate of more than 50%. 7 Conversely, breast tumor xenografts derived from patient biopsies are particularly difficult to obtain, as reported by several teams (3). Breast tumors have one of the lowest tumor take rates in immunodeficient mice.…”

Role of Human Pregnane X Receptor in Tamoxifen- and 4-Hydroxytamoxifen-Mediated CYP3A4 Induction in Primary Human Hepatocytes and LS174T Cells

“…Following activation of Amplitaq Gold DNA polymerase, cDNA was amplified using cycling conditions: 10 min of initial denaturation at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C for primer annealing and extension. Primers: PXR (NM_003889) forward: 5Ј-GCA TCATCA GCT TTG CCA AAG-3Ј, reverse: 5Ј-CCG CGT TGA ACA CTG TGT TG-3Ј; HNF4␣ (NM_178849) forward: 5Ј-AGC CTG CCC TCC ATC AAT G-3Ј, reverse: 5Ј-CTC ACA CAC ATC TGC GAT GCT-3Ј; retinoid X receptor (RXR) ␣ (NM_002957) (Haugen et al, 2004) forward: 5Ј-GAG GCC TAC TGC AAG CAC AAG-3Ј, reverse: 5Ј-CAG GCG GAG CAA GAG CTT AG-3Ј; GR␣ (NM_000176) (Raddatz et al, 2004) forward: 5Ј-CAA AAC TCT TGG ATT CTA TGC ATG AA-3Ј, reverse: 5Ј-TTG GAA GCA ATA GTT AAG GAG ATT TTC-3Ј; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM_002046) forward: 5Ј-GAA GGT GAA GGT CGG AGT C-3Ј, reverse: 5Ј-GAA GAT GGT GAT GGG ATT TC-3Ј; CYP3A4 (D11131) forward: 5Ј-CTT CAT CCA ATG GAC TGC ATA AAT-3Ј, reverse 5Ј-TCC CAA GTA TAA CAC TCT ACA CAG ACA A-3Ј (Bowen et al, 2000); and ER␣ (NM_000125) forward: 5Ј-CCA CCA ACC AGT GCA CCA TT-3Ј, reverse: 5Ј-GGT CTT TTC GTA TCC CAC CTT TC-3Ј (de Cremoux et al, 2004).…”