• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5815-5818
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• 2014

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.

• Journal of dental hygiene science
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• v.17 no.5
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• pp.433-438
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• 2017

Relative to its incidence, oral cancer has serious negative social effects. The exact causes of oral cancer have not been clarified, but many studies have implicated smoking and drinking. However, the fundamental mechanism of oral cancer causation has yet to be elucidated. Lysophosphatidic acid (LPA) augments epithelial mesenchymal transition (EMT) and development of various cancer cells. However, a detailed mechanistic explanation for LPA-induced EMT and the effects of EMT-promoting conditions on oral squamous cell carcinoma development remain elusive. In the present study, a quantitative reverse transcription polymerase chain reaction was used to analyze TWIST1, Slug, E-cadherin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript expression. Immunoblotting was used to analyze TWIST1, Slug, E-cadherin, and GAPDH protein expression. siRNAs were used to silence TWIST1 and Slug transcript expression. A matrigel-coated in vitro invasion insert was used to analyze oral cancer cell invasion. The results of the present study show that the expression levels of TWIST1 and Slug, which are EMT factors, were increased by LPA treatment in YD-10B oral squamous cell carcinoma. Conversely, E-cadherin expression was significantly reduced. In addition, transfection of the cells with TWIST1 and Slug siRNA strongly inhibited LPA-induced oral cancer cell invasion. The present study shows that TWIST1 and Slug mediate LPA-induced oral cancer cell EMT and invasiveness. The present study confirmed the mechanism by which LPA promotes oral cancer cell development, with TWIST1 and Slug providing novel biomarkers and promising therapeutic targets for oral cancer cell development.

• Food Science and Preservation
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• v.14 no.6
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• pp.688-694
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• 2007

To evaluate the human risk of long-term intake of genetically modified (GM) rice, we carried out RT-PCR of housekeeping genes. Housekeeping genes, which show highly uniform expression in living organisms during various stages of development and under different environmental conditions, were normalized by RT-PCR. We assessed the expression of 10 common housekeeping genes (18s rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-$1{\alpha}$, ${\beta}$-TUB, GAPDH, ${\beta}$-actin, B2m, G6pd2, Gyk, Gus, Hprt, Cyclophlin A, Tfrc, ${\alpha}$-tubulin and RPL13A) in the liver, stomach, small intestine, large intestine, kidney and spleen of mice fed GM or non-GM rice. We found no significant differences in the expression of housekeeping genes between the two groups of mice.

• Biomedical Science Letters
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• v.29 no.4
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• pp.220-230
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• 2023

Identification of the pathogens causing infection is important in terms of patient's health management and infection control. Synovial fluids could be used as clinical samples to detect causative pathogens of prosthetic joint infections (PJIs) using molecular diagnostic assays, therefore, normalization and validation of clinical samples are necessary. Microbial culture is considered the gold standard for all infections, including PJIs. Recently, molecular diagnostic methods have been developed to overcome the limitation of microbial culture. Therefore, guideline for validating clinical samples to provide reliable results of molecular diagnostic assays for infectious diseases is required in clinical field. The present study aimed to develop an accurate validating method of synovial fluid clinical samples using GAPDH gene as an internal control to perform the quantitative PCR TaqMan probe assay to detect pathogens causing PJIs.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.39-46
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• 2016

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (${\beta}-actin$), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

• ALGAE
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• v.31 no.2
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• pp.167-174
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• 2016

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1507-1513
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• 2015

Pemetrexed is an antifolate agent which has been used for treating malignant pleural mesothelioma and non small lung cancer in the clinic as a chemotherapeutic agent. In this study, pemetrexed inhibited cell growth and induced G1 phase arrest in the A549 cell line. To explore the molecular mechanisms of pemetrexed involved in cell growth, we used a two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomics approach to analyze proteins changed in A549 cells treated with pemetrexed. As a result, twenty differentially expressed proteins were identified by ESI-Q-TOF MS/MS analysis in A549 cells incubated with pemetrexed compared with non-treated A549 cells. Three key proteins (GAPDH, HSPB1 and EIF4E) changed in pemetrexed treated A549 cells were validated by Western blotting. Accumulation of GAPDH and decrease of HSPB1 and EIF4E which induce apoptosis through inhibiting phosphorylation of Akt were noted. Expression of p-Akt in A549 cells treated with pemetrexed was reduced. Thus, pemetrexed induced apoptosis in A549 cells through inhibiting the Akt pathway.

• Food Science and Preservation
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• v.15 no.4
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• pp.562-567
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• 2008

To develop human risk assessment protocols, we explored housekeeping gene and cytokine expression in mouse spleen cells using Rt-PCR. We normalized housekeeping gene expression by RT-PCR; gene expression was highly uniform in potato leafs and mouse spleen cells. We measured the expression of frequently used housekeeping genes, such as those encoding APRT, $\beta$-tubulin, Actin, Hsp 20.2, Cyclophilin, 18S RNA, Efla, Tbp, GAPDH, $\beta$-actin, Tuba2, Hprt, Cyclophlin A, Tfrc, and RPL13A in mice fed GM or non-GM potatoes. Housekeeping gene expression did not show any significant differences between GM and non-GM potato-fed mice. The murine model of potato-fed mice did not express IL-4 and IL-13 at a significant levels.

• Food Science and Preservation
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• v.15 no.1
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• pp.84-87
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• 2008

We used RT-PCR to measure housekeeping gene expression in mice fed GM and non-GM cabbage, in an effort to evaluate the risk of GM food to humans. After normalization of housekeeping gene levels, highly uniform expression may be seen in many organisms during various stages of development and under different environmental conditions. We assessed the expression of four genes in Chinese cabbage; these were Profilin, Tubulin-alpha (Tub-1), Heat-shock protein (Bchsp 17.6), and Ubiquitin conjugating enzyme (UBE). We measured the expression of four well-known housekeeping genes in mice: ${\beta}$-actin, (${\beta}$-act), ${\beta}$-2-microglobulin(B2m), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ${\beta}$-glucuronidase (Gus). Gene expression was measured in liver, stomach, small intestine, large intestine, kidney, and spleen of mice fed GM or non-GM cabbage. No significant expression differences were found.

• Journal of Preventive Medicine and Public Health
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• v.45 no.4
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• pp.235-243
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• 2012

Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.