sybr-green-i and Breast-Neoplasms

The aims of this study were to detect \ CCND1\ , \ C-MYC\ , and \ FGFR1\ amplification using qPCR, confirmation with \ FISH, and to further assess their clinicopathological relevance.. Thirty-five breast tumor samples were analyzed for amplification of the selected genes using modified SYBR \ Green qPCR. The accuracy of the qPCR was assessed by FISH as a gold-standard method.. CCND1\ , \ C-MYC\ , and \ FGFR1 \ amplifications were observed in 34.28%, 28.57%, and 17.14% of the 35 samples, respectively.\ qPCR results were significantly confirmed by FISH and qPCR and FISH showed excellent correlation (P = 0.000). \ CCND1\ amplification \ with tumor stage (P = 0.044), positive metastatic status (P = 0.042), positive family history (P = 0.042), and \ C-MYC\ status (P = 0.005); \ C-MYC\ amplification with tumor size (P = 0.021), tumor grade (P = 0.018), tumor stage (P = 0.032), and \ FGFR1\ status (P < 0.000); and \ FGFR1\ amplification with tumor size (P = 0.041) and positive \ ER\ status (P = 0.042) were statistically associated.. Our findings revealed that the applied qPCR approach could precisely quantify the relative gene copy number. More \ studies with a larger sample size are suggested to confirm the clinicopathological value of \ CCND1\ , \ C-MYC\ , and \ FGFR1\ amplification.

Topics: Adolescent; Adult; Benzothiazoles; Breast Neoplasms; Carcinoma, Ductal, Breast; Cyclin D1; Diamines; DNA-Binding Proteins; Female; Gene Amplification; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Middle Aged; Neoplasm Grading; Neoplasm Staging; Organic Chemicals; Quinolines; Real-Time Polymerase Chain Reaction; Receptor, Fibroblast Growth Factor, Type 1; Reproducibility of Results; Transcription Factors; Young Adult

Growing evidence suggests microRNAs (miRNAs) have an important role in tumorigenesis. MicroRNA-21 (miR-21) is up-regulated in many malignant tumors, including breast cancer. Its association with clinicopathologic features and expression of PTEN (phosphatase and tensin homolog deleted on chromosome 10), one of its target genes, in breast cancer has not been reported systematically. To further determine the potential involvement of miR-21 in breast cancer, we have evaluated the expression level of miR-21 by stem-loop real-time RT-PCR based on SYBR-Green I in human invasive ductal carcinoma of the breast, and we have correlated the results with clinicopathologic features and PTEN protein expression. Matched non-tumor and tumor tissues of 40 human invasive ductal carcinoma of the breast were analyzed for miR-21 expression by stem-loop real-time RT-PCR based on SYBR-Green I. Immunohistochemistry (IHC) was used to estimate PTEN expression in tumor tissue. The expression levels of miR-21 were correlated with PTEN and commonly used clinicopathologic features of breast cancer. The stem-loop real-time RT-PCR based on SYBR-Green I was sensitive and specific enough to detect miR-21. Expression levels of miR-21 were significantly higher in tumor tissues than the levels in matched non-tumor tissues (P=0.000). Expression of miR-21 was negatively correlated with expression of PTEN (P=0.013). Up-regulated miR-21 expression was associated with lymph node positivity (P=0.01), higher proliferation index (ki67>10%) (P=0.03) and advanced breast cancer TNM clinical stage (P=0.021). These findings suggest that PTEN is possibly one of the targets of miR-21 in breast cancer and high expression of mir-21 indicates a more aggressive phenotype.

Topics: Benzothiazoles; Breast Neoplasms; Carcinoma, Ductal, Breast; Diamines; Female; Humans; Immunohistochemistry; MicroRNAs; Neoplasm Staging; Organic Chemicals; PTEN Phosphohydrolase; Quinolines; Reverse Transcriptase Polymerase Chain Reaction

We compared two technologies of real-time PCR (with the use of fluorescent SYBR Green I dye and specific TaqMan probe) for quantification of the dose of her2 gene in breast tumors. The maximum increase in the gene dose in TaqMan and SYBR Green I analyses was 10- and 5-fold, respectively. In was found that TaqMan and SYBR Green I technologies allow detection of the matrix in amounts corresponding to 1-100 and 2.5-40.0 ng genomic DNA, respectively. Tenfold increase in the gene dose leads to incorrect evaluation of multiplication ratio in the SYBR Green I analysis. These results suggest that TaqMan technology is more preferable for correct evaluation of her2 gene dose.

Topics: Benzothiazoles; Breast Neoplasms; Diamines; Female; Fluorescent Dyes; Gene Dosage; Humans; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Receptor, ErbB-2

Real-time reverse transcriptase polymerase chain reaction is recognized as a highly sensitive and specific method for quantification of mRNA expression. SYBR green I dye simplifies the experimental design but introduces the need for specific controls to maintain high specificity. Due to this increased sensitivity, standards that may have been acceptable for normalization of less sensitive methods have been shown to vary considerably among cell lines, tissues, proliferative states, treatments, and developmental conditions and by degree of cancer progression. It has become evident that determination of suitable normalization standards is a requirement for the use of this method as it is applied toward any new experimental model. We have assessed the suitability of a number of commonly used standards for the normalization of mRNAs among a set of human breast cancer cell lines of increasing metastatic potential and have determined that 18S rRNA and beta-actin (ACTB) mRNA are both suitable for this purpose, with each having some limitations. 18S rRNA varies less among the cell lines but has a higher degree of random variability, while ACTB mRNA varies more among cell lines but has a lower degree of random variation.

Topics: Actins; Benzothiazoles; Breast Neoplasms; Cell Line, Tumor; Diamines; Female; Humans; Organic Chemicals; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; RNA, Ribosomal, 18S; Sensitivity and Specificity

cDNA microarray analysis is highly useful for monitoring genome-wide changes in gene expression that occur in biological processes. Current standards require that microarray observations be verified by quantitative (Q)-PCR or other techniques. Few studies have optimized Q-PCR for verification of microarray findings. The current study assessed several variables affecting Q-PCR fidelity, including RNA extraction methods, mRNA enrichment, primers for reverse transcription, and cDNA amplification detection methods. Also assessed was the choice of reference gene on which other gene expression changes are based. The RNA for ribosomal protein S28 was found to be ideal for this purpose, with minimal variance in expression among isogenic drug-resistant cell lines. We also found that oligo (dT) primers were superior to random hexamers and that RNA extracted by the RNeasy method gave consistent S28 gene amplification without the need for mRNA enrichment, particularly when TaqMan probes were used. Nevertheless, sensitivity was sufficiently high with SYBR Green I that it was the preferred, least costly method for amplification product detection, even for low-abundance transcripts. Using the optimal method, 91-95% of the differences in gene expression identified between the cell lines by cDNA microarray analysis could be confirmed by Q-PCR, significantly superior to previously described methods.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents, Phytogenic; Benzothiazoles; Breast Neoplasms; Cell Line, Tumor; Diamines; DNA, Complementary; Doxorubicin; Drug Resistance, Multiple; Female; Gene Amplification; Gene Expression; Humans; Microarray Analysis; Organic Chemicals; Paclitaxel; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger

Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce 'primer dimer' formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.

Topics: Benzothiazoles; Breast Neoplasms; Cell Line, Tumor; Diamines; DNA Primers; Fluorescent Dyes; Gene Expression Profiling; Humans; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm

Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays. Cells were inoculated on the CAM of 9-day-old chick embryos and the membrane at the opposite side of the egg was recovered after 48 h incubation. To measure intravasation ability, we calculated the amount of human DNA in each CAM sample by real-time PCR of Alu sequences and SYBR Green 1 fluorescence detection. This analysis showed a detection limit of 1 human cell per 10(5) total cells, and we were able to distinguish between tumor cells of distinct invasive capacity. This assay has several advantages over current methods to measure intravasation ability, including the elimination of post-PCR analysis, sensitivity and easy scale-up of sample numbers.

Topics: Adenocarcinoma; Allantois; Alu Elements; Animals; Benzothiazoles; Breast Neoplasms; Chick Embryo; Chorion; Computer Systems; Diamines; DNA, Neoplasm; Ethidium; Fibroblasts; Fluorescent Dyes; Humans; Neoplasm Invasiveness; Organic Chemicals; Phenotype; Polymerase Chain Reaction; Quinolines; Tumor Cells, Cultured; Urinary Bladder Neoplasms