Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: Comparison of real-time reverse transcription-PCR vs. Relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products

Agnes Juhasz, Paul Frankel, Catherine Cheng, Hector Rivera, Reena Vishwanath, Alice Chiu, Kim Margolin, Yun Yen, Edward M. Newman, Tim Synold, Sharon Wilczynski, Heinz Josef Lenz, David Gandara, Kathy S. Albain, Jeffrey Longmate, James H. Doroshow

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The solid tumor mRNA expression of genes related to the mechanism of action of certain antineoplastic agents is often predictive of clinical efficacy. We report here on the development of a rapid and practical real-time RT-PCR method to quantify genetic expression in solid tumors. The genes examined are related to the intracellular pharmacology of gemcitabine and cisplatin, two drugs that are used in the treatment of several types of advanced cancer. We evaluated target gene mRNA levels from breast tumor samples using two quantitative RT-PCR methods: 1) an improved relative RT-PCR method using fluorescence-labeled primers, automated PCR set up, and GeneScan® analysis software; and 2) real-time RT-PCR with redesigned primers using an ABI 7900HT instrument, with additional postprocessing of the data to adjust for efficiency differences across the target genes. Using these methods, we quantified mRNA expression levels of deoxycytidine kinase (dCK), deoxycytidylate deaminase (dCDA), the M1 and M2 subunits of ribonucleotide reductase (RRM1, RRM2), and excision cross complementation group 1 (ERCC1) in 35 human "fresh" frozen breast cancer biopsies. While both assay methods were substantially more rapid than traditional RT-PCR, real-time RT-PCR appeared to be superior to the amplification end-point measurement in terms of precision and high throughput, even when a DNA sequencer was used to assess fluorescence-labeled PCR products. This reproducible, highly sensitive real-time RT-PCR method for the detection and quantification of the mRNAs for dCK, dCDA, RRM1, RRM2, and ERCC1 in human breast cancer biopsies appears to be more informative and less time-consuming than either classical radioisotope-dependent RT-PCR or the technique utilizing GeneScan® analysis described herein. By allowing the measurement of intratumoral target gene expression, these new methods may prove useful in predicting the clinical utility of gemcitabine- and platinum-containing chemotherapy programs in patients with solid tumors.

Original languageEnglish
Pages (from-to)184-194
Number of pages11
JournalJournal of Clinical Laboratory Analysis
Volume17
Issue number5
DOIs
Publication statusPublished - Sep 26 2003
Externally publishedYes

Fingerprint

Biopsy
Transcription
gemcitabine
Reverse Transcription
Tumors
DCMP Deaminase
Genes
Deoxycytidine Kinase
Breast Neoplasms
Gene Expression
Polymerase Chain Reaction
Messenger RNA
DNA
Real-Time Polymerase Chain Reaction
Fluorescence
Chemotherapy
Platinum
Gene expression
Radioisotopes
Neoplasms

Keywords

  • Breast cancer biopsy
  • Gemcitabine and cisplatin
  • Gene expression
  • Real-time RT-PCR

ASJC Scopus subject areas

  • Immunology and Allergy
  • Hematology
  • Public Health, Environmental and Occupational Health
  • Clinical Biochemistry
  • Medical Laboratory Technology
  • Biochemistry, medical
  • Microbiology (medical)

Cite this

Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies : Comparison of real-time reverse transcription-PCR vs. Relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products. / Juhasz, Agnes; Frankel, Paul; Cheng, Catherine; Rivera, Hector; Vishwanath, Reena; Chiu, Alice; Margolin, Kim; Yen, Yun; Newman, Edward M.; Synold, Tim; Wilczynski, Sharon; Lenz, Heinz Josef; Gandara, David; Albain, Kathy S.; Longmate, Jeffrey; Doroshow, James H.

In: Journal of Clinical Laboratory Analysis, Vol. 17, No. 5, 26.09.2003, p. 184-194.

Research output: Contribution to journalArticle

Juhasz, A, Frankel, P, Cheng, C, Rivera, H, Vishwanath, R, Chiu, A, Margolin, K, Yen, Y, Newman, EM, Synold, T, Wilczynski, S, Lenz, HJ, Gandara, D, Albain, KS, Longmate, J & Doroshow, JH 2003, 'Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies: Comparison of real-time reverse transcription-PCR vs. Relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products', Journal of Clinical Laboratory Analysis, vol. 17, no. 5, pp. 184-194. https://doi.org/10.1002/jcla.10091
Juhasz, Agnes ; Frankel, Paul ; Cheng, Catherine ; Rivera, Hector ; Vishwanath, Reena ; Chiu, Alice ; Margolin, Kim ; Yen, Yun ; Newman, Edward M. ; Synold, Tim ; Wilczynski, Sharon ; Lenz, Heinz Josef ; Gandara, David ; Albain, Kathy S. ; Longmate, Jeffrey ; Doroshow, James H. / Quantification of chemotherapeutic target gene mRNA expression in human breast cancer biopsies : Comparison of real-time reverse transcription-PCR vs. Relative quantification reverse transcription-PCR utilizing DNA sequencer analysis of PCR products. In: Journal of Clinical Laboratory Analysis. 2003 ; Vol. 17, No. 5. pp. 184-194.
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