Scanning copy number and gene expression on the 16p13.3‐13.2 chromosomal region by the systematic multiplex polymerase chain reaction and reverse transcription‐polymerase chain reaction methods

Yamamoto, Miyako; Ahn, Ray Hyungjoo; Yamamoto, Fumiichiro

doi:10.1002/elps.200500875

Cited by 3 publications

(5 citation statements)

References 15 publications

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“…Using a model SM (RT-)PCR system that contained genes from autosomes and the X chromosome, we previously demonstrated that less than a twofold difference in copy number could be detected by SM PCR [11]. In the present study we applied SM PCR and SM RT-PCR to examine the changes in copy number and expression of more than a hundred of genes in breast and prostate tumors and cancer cell lines.…”

Section: Discussionmentioning

confidence: 96%

“…Using control genomic DNA template, the optimal primer concentrations were determined to unify band intensity as previously described [8][9][10][11]. Out of 140 genes on the chromosomal region of 18q21-qter, 13 genes failed to amplify specifically and were excluded from the system.…”

Section: Sm Pcr and Sm Rt-pcr Analyses Of The Genes On The Chromosomamentioning

confidence: 99%

“…The detailed experimental procedures to establish the SM (RT-)PCR system have been previously described [8][9][10][11]. Briefly, the genes were categorized into groups of approximately ten genes, and the concentrations of PCR primers in multiplex reactions were optimized to amplify different sizes of DNA fragments in single exons at similar band intensities using genomic DNA from normal human tissues as a control.…”

Section: Sm Pcr and Sm Rt-pcr Experiments To Measurementioning

confidence: 99%

“…There are not many genes in the 18q21-qter region, approximately 140, so we used the systematic multiplex (SM) PCR and SM reverse transcription (RT)-PCR methods, which we developed for semiquantitative analyses of copy number and gene expression [8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

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Scanning copy number and gene expression on the 18q21‐qter chromosomal region by the systematic multiplex PCR and reverse transcription‐PCR methods

Yamamoto

2007

Electrophoresis

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We examined differences in copy number and expression of 127 genes located on the 18q21-qter chromosomal region of the breast and prostate cancer cell lines, using the systematic multiplex PCR and reverse transcription-PCR (SM PCR and SM RT-PCR) methods that we developed. Semi-quantitative data were obtained that were comparable in quality, but not in quantity, to data from DNA microarray hybridization analysis. In the chromosomal region where losses are frequent in breast, prostate, and other cancers, we detected a homozygous deletion of the SMAD4 gene in the MDA-MB-468 breast cancer cell line. We also observed partial or entire loss of expression in genes such as CCBE1, CCDC11, CD226, NP_115536.1, NP_689683.2, RNF152, SERPINB8, and TCF4 in certain breast and/or prostate cancer cell lines. An increase in gene expression was rare, but found with the transcription factor ONECUT2 gene in all of the cancer cell lines examined. Real-time qRT-PCR experiments confirmed these SM RT-PCR results. Further analysis of clinical specimens of breast cancer by real-time qRT-PCR demonstrated that the gene expression of CCBE1, TCF4, NP_115536.1, and NP_689683.2 was downregulated in the majority of clinical cases of breast cancer.

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Section: Discussionmentioning

confidence: 96%

Section: Sm Pcr and Sm Rt-pcr Analyses Of The Genes On The Chromosomamentioning

confidence: 99%

Section: Sm Pcr and Sm Rt-pcr Experiments To Measurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Scanning copy number and gene expression on the 18q21‐qter chromosomal region by the systematic multiplex PCR and reverse transcription‐PCR methods

Yamamoto

2007

Electrophoresis

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“…We prepared cDNA by reverse-transcription using oligo dT primers and the Advantage RT-for-PCR Kit (BD Biosciences-Clontech). We followed the SM RT-PCR experimental protocols described previously [29], [50], [51], [52]. Complementary DNA samples were used as templates to examine gene expression.…”

Section: Methodsmentioning

confidence: 99%

Rare and Frequent Promoter Methylation, Respectively, of TSHZ2 and 3 Genes That Are Both Downregulated in Expression in Breast and Prostate Cancers

et al. 2011

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BackgroundNeoplastic cells harbor both hypomethylated and hypermethylated regions of DNA. Whereas hypomethylation is found mainly in repeat sequences, regional hypermethylation has been linked to the transcriptional silencing of certain tumor suppressor genes. We attempted to search for candidate genes involved in breast/prostate carcinogenesis, using the criteria that they should be expressed in primary cultures of normal breast/prostate epithelial cells but are frequently downregulated in breast/prostate cancer cell lines and that their promoters are hypermethylated.Methodology/Principal FindingsWe identified several dozens of candidates among 194 homeobox and related genes using Systematic Multiplex RT-PCR and among 23,000 known genes and 23,000 other expressed sequences in the human genome by DNA microarray hybridization. An additional examination, by real-time qRT-PCR of clinical specimens of breast cancer, further narrowed the list of the candidates. Among them, the most frequently downregulated genes in tumors were NP_775756 and ZNF537, from the homeobox gene search and the genome-wide search, respectively. To our surprise, we later discovered that these genes belong to the same gene family, the 3-member Teashirt family, bearing the new names of TSHZ2 and TSHZ3. We subsequently determined the methylation status of their gene promoters. The TSHZ3 gene promoter was found to be methylated in all the breast/prostate cancer cell lines and some of the breast cancer clinical specimens analyzed. The TSHZ2 gene promoter, on the other hand, was unmethylated except for the MDA-MB-231 breast cancer cell line. The TSHZ1 gene was always expressed, and its promoter was unmethylated in all cases.Conclusions/SignificanceTSHZ2 and TSHZ3 genes turned out to be the most interesting candidates for novel tumor suppressor genes. Expression of both genes is downregulated. However, differential promoter methylation suggests the existence of distinctive mechanisms of transcriptional inactivation for these genes.

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Identification of Genes That Exhibit Changes in Expression on the 8p Chromosomal Arm by the Systematic Multiplex RT-PCR (SM RT-PCR) and DNA Microarray Hybridization Methods

Yamamoto¹,

Yamamoto²

2008

gene expr

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Losses of the p-arm of chromosome 8 are frequently observed in breast, prostate, and other types of cancers. Using the Systematic Multiplex RT-PCR (SM RT-PCR) method and the DNA microarray hybridization method, we examined the expression of 273 genes located on the p-arm of chromosome 8 in five breast and three prostate human cancer cell lines. We observed frequent decreases in expression of two dozen genes and increases in expression of several genes on this chromosomal arm. These changes in gene expression of the cell lines were later confirmed by real-time qRT-PCR. Additionally and more importantly, we found that a number of these variations were also observed in the majority of clinical cases of breast cancer we examined. These included downregulation of the MYOM2, NP_859074, NP_001034551, NRG1, PHYIP (PHYHIP), Q7Z2R7, SFRP1, and SOX7 genes, and upregulation of the ESCO2, NP_115712 (GINS4), Q6P464, and TOPK (PBK) genes.

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Scanning copy number and gene expression on the 16p13.3‐13.2 chromosomal region by the systematic multiplex polymerase chain reaction and reverse transcription‐polymerase chain reaction methods

Cited by 3 publications

References 15 publications

Scanning copy number and gene expression on the 18q21‐qter chromosomal region by the systematic multiplex PCR and reverse transcription‐PCR methods

Scanning copy number and gene expression on the 18q21‐qter chromosomal region by the systematic multiplex PCR and reverse transcription‐PCR methods

Rare and Frequent Promoter Methylation, Respectively, of TSHZ2 and 3 Genes That Are Both Downregulated in Expression in Breast and Prostate Cancers

Identification of Genes That Exhibit Changes in Expression on the 8p Chromosomal Arm by the Systematic Multiplex RT-PCR (SM RT-PCR) and DNA Microarray Hybridization Methods

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