• Asian-Australasian Journal of Animal Sciences
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• v.26 no.7
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• pp.921-929
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• 2013

Huoyan goose is a Chinese local breed famous for its higher laying performance, but the problems of variety degeneration have emerged recently, especially a decrease in the number of eggs laid. In order to better understand the molecular mechanism that underlies egg laying in Huoyan geese, gene profiles in the pituitary gland of Huoyan geese taken during the laying period and ceased period were investigated using the suppression subtractive hybridization (SSH) method. Total RNA was extracted from pituitary glands of ceased period and laying period geese. The cDNA in the pituitary glands of ceased geese was subtracted from the cDNA in the pituitary glands of laying geese (forward subtraction); the reverse subtraction was also performed. After sequencing and annotation, a total of 30 and 24 up and down-regulated genes were obtained from the forward and reverse SSH libraries, respectively. These genes mostly related to biosynthetic process, cellular nitrogen compound metabolic process, transport, cell differentiation, cellular protein modification process, signal transduction, small molecule metabolic process. Furthermore, eleven genes were selected for further analyses by quantitative real-time PCR (qRT-PCR). The qRT-PCR results for the most part were consistent with the SSH results. Among these genes, Synaptotagmin-1 (SYT1) and Stathmin-2 (STMN2) were substantially over-expressed in laying period compared to ceased period. These results could serve as an important reference for elucidating the molecular mechanism of higher laying performance in Huoyan geese.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.132-137
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• 2008

Differential gene expression profiling was carried out in the hepatic tissue of medaka fish, Oryzias latipes, after exposure to an organophosphorus pesticide (OPP), Iprobenfos (IBP), a widely used pesticide in agri- and fish-culture, using a medaka cDNA micro array. Twenty six kinds of differentially expressed candidate genes, with 15 and 11 induced and repressed in their gene expressions, respectively, were associated with cytoskeleton (3.8%), development (7.7%), immune (7.7%), metabolism (30.8%), nucleic acid/protein binding (42.3%) and reproduction (7.7%). Of these genes, changes at the transcription level of five were re-evaluated by real-time quantitative PCR (qRT-PCR). Considering the known function of authentic genes, the effects of IBP on the biological activity and pathological aspects in medaka fish were discussed. The identified genes could be used as molecular biomarkers for biological responses to OPPs contamination in an aquatic environment.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.112-121
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• 2017

Edwardsiella tarda is a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors of E. tarda. Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene in E. tarda, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (recA, uup, rpoB, rho, topA, gyrA, groEL, rpoD, and 16S rRNA) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that 16S rRNA was more stable than the other genes at different culture growth phases. However, at the same culture time, topA was identified as the reference gene under the conditions of different strains, different culture media, and infection, whereas gyrA was identified under the condition of different temperatures. Thus, in experiments, the expression of gapA and fbaA in E. tarda was analyzed by RT-qPCR using 16S rRNA, recA, and uup as the reference genes. The results showed that 16S rRNA was the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1280-1286
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• 2011

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at $3.6{\times}10^6$ CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above $10^5$/ml for approx. 10 days, after which cell numbers decreased to levels of $10^3$ CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. $1{\times}10^2$ CFU/ml was detected. The minimum detection level for quantitative PCR in this study was $1{\times}10^2$ to $1{\times}10^3$ CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.15 no.1
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• pp.38-43
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• 2012

Purpose: Epstein-Barr virus (EBV) hepatitis is a usually asymptomatic and self-limiting disease in immunocompetent patients. However, the range of severity is wide, and the serological diagnosis is typically difficult until the convalescent phase. Thus, we examined the value of plasma EBV DNA real-time quantitative polymerase chain reaction (RT-qPCR) in EBV hepatitis for the timely diagnosis and the relationship between EBV viral load and clinical severity. Methods: Sixty samples were confirmed as having EBV infection by RT-qPCR with the EBV BALF5 gene sequence. We examined the clinical characteristics of EBV hepatitis by reviewing medical records. Results: The median total duration of fever was 8 days (range: 0-13 days). The mean peak value of aspartate aminotransferase (AST) was $241{\pm}214$ U/L, and the mean peak value of alanine aminotransferase (ALT) was $298{\pm}312$ U/L. There was no correlation between the serum levels of liver enzyme and plasma EBV DNA titer ($p$=0.1) or between median total duration of fever and EBV DNA titer ($p$=0.056). The median age of the EBV VCA IgM-negative group was lower compared with the EBV VCA IgM-positive group in EBV hepatitis (2 years vs. 6 years, $p$=0.0009). Conclusion: The severity of EBV hepatitis does not correlate with circulating EBV DNA load according to our data. Furthermore, we suggest that plasma EBV PCR may be valuable in young infants in whom the results of serology test for EBV infection commonly are negative.

• Molecules and Cells
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• v.28 no.2
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• pp.99-103
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• 2009

Human endogenous retroviruses (HERVs) have been implicated in the pathogenesis of several human diseases as multi-copy members in the human genome. Their gene expression profiling could provide us with important insights into the pathogenic relationship between HERVs and cancer. In this study, we have evaluated the genomic structure and quantitatively determined the expression patterns in the env gene of a variety of HERV family members located on six specific loci by the RetroTector 10 program, as well as real-time RT-PCR amplification. The env gene transcripts evidenced significant differences in the human tumor/normal adjacent tissues (colon, liver, uterus, lung and testis). As compared to the adjacent normal tissues, high levels of expression were noted in testis tumor tissues for HERV-K, in liver and lung tumor tissues for HERV-R, in liver, lung, and testis tumor tissues for HERV-H, and in colon and liver tumor tissues for HERV-P. These data warrant further studies with larger groups of patients to develop biomarkers for specific human cancers.

• Development and Reproduction
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• v.7 no.2
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• pp.127-136
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• 2003

The present study was conducted to investigate gene expression profile of mouse ovaries during the primordial-primary follicle transition. We isolated total RNA from mouse ovaries at day1(contains only primordial follicles) and day5(contains both primordial and primary follicles) and synthesized cDNA using annealing control primers(ACP, Seegene, Inc., Seoul, Korea). Using 80 different ACPs for PCR, we cloned, sequenced, and analyzed identities of 41 differentially expressed genes(DEGs). According to BLAST analysis, sequences of 33 clones significantly matched database entries, 4 clones were novel, and 4 clones were ESTs. We selected 8 DEGs with interesting functions, Anx11 and Pepp2-Pending highly expressed in day1 ovary, while Apg3/Autlp-like, BPOZ, Ches1, Kcmf1, NHE3, Nid2, Ninj1, SENP3, Suil-rsl, and TIAP/m-survivin highly expressed in days ovary, and confirmed their different expression between day1 ovaries and days ovaries using semi-quantitative RT-PCR. There was no false positive result. Using in situ hybridization, we found that almost all of genes studied were expressed in the oocyte from primordial follicle stage but expression decreased from primary follicle stage. Meanwhile their expression was increased in cuboidal granulosa cells. Different expression of BPOZ and TIAP/m-survivin between primordial and primary follicles was confirmed by using laser capture microdissection followed by real-time PCR BPOZ and TIAP/m-survivin expressed 4.5 and 3.4 fold higher in primary than primordial follicles, respectively. List of genes obtained from the present study will provide insights for the study of mechanism regulating primordial-primary follicle transition.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.8
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• pp.987-992
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• 2010

This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5879-5882
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• 2012

Breast cancer is the leading cause of cancer deaths among women worldwide, including Thailand. In the present study, the differential mRNA expression of SVEP1, LPHN3, KLB, ITGA7, SEMA3G, TNS1 and MMP13 genes was examined in breast cancer using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Among these genes, increased LPHN3 and MMP13 mRNA expression levels correlated with axillary-node metastasis (P=0.02). Multiple logistic regression analysis revealed that LPHN3 and MMP13 mRNA expression is significantly associated with axillary node status in breast cancer (P=0.04).

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2305-2309
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• 2012

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin ($IC_{50}$ 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 ${\mu}g$ protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the $T_m$ telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.