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

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.

Regulation of DNA repair gene expression in human cancer cell lines

“…This is the first study to examine XPA or XPF expression using real‐time quantitative PCR. ERCC1 expression has recently been examined in colorectal tumors [Shirota et al, 2001] non‐small cell lung tumors [Lord et al, 2002], breast tumors [Juhasz et al, 2003], and a human leukemia cell line [Galm et al, 2002] by real‐time quantitative PCR.…”

Systemic delivery of siRNA nanoparticles targeting RRM2 suppresses head and neck tumor growth

“…Tumor RNA samples were reverse-transcribed using SuperScriptIII reverse transcriptase. 2 μl of prepared sample cDNA was used for triplicate Taqman real time-PCR as described elsewhere [32]. RRM2 levels were normalized to Tata Binding Protein (TBP) levels within the same sample.…”

Down-regulation of PRKCB1 expression in Han Chinese patients with subsyndromal symptomatic depression

“…We used the DDCt method (Juhasz et al, 2003) to calculate the fold change of gene expression. The DCt-value of each target gene in each sample was obtained by subtracting the average GAPDH Ctvalue from the average Ct-value of each sample.…”