TaqMan Real-time Quantitative PCR Research Articles

Expression of Src Suppressed C Kinase Substrate in Rat Neural Tissues During Inflammation

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney etc., indicating a possible role of SSeCKS in inflammatory process. However, the expression and biological function of SSeCKS during neuronal inflammation remains to be elucidated, so we established an inflammatory model injected with LPS to investigate the gene expression patterns of SSeCKS in neural tissues by using TaqMan quantitative real-time PCR and immunohistochemistry in rat. Real-time PCR showed that LPS stimulated the expression of SSeCKS mRNA in a dose- and time-dependent manner in sciatic nerves, spinal cords and dorsal root ganglions. Immunohistochemistry showed that SSeCKS colocalized with nerve fibers in sciatic nerve after LPS administration, but there was no colocalization between SSeCKS and Schwann cells. In addition, SSeCKS colocalized with neurons which existed in dorsal root ganglions and spinal cords. These findings indicated that SSeCKS might play some important roles in sciatic nerve fibers and neurons in spinal cords and dorsal root ganglions after LPS injection.

Effects of antidepressant drug exposure on gene expression in the developing cerebral cortex

To clarify the basis of limited responses in children and adolescents to antidepressant treatments considered standard in the treatment of adult major depressive disorder, juvenile Sprague-Dawley rats were subjected to 21-day treatment with dissimilar antidepressant drugs fluoxetine, imipramine, or vehicle control. Total RNA was extracted from brain frontal cortices and hybridized to the Affymetrix 230.2 chip. A total of 18 microarrays were analyzed (i.e., six biological replicates in three treatment groups). Transcripts identified were validated using Taqman real-time quantitative PCR methodology, and the relative expression of each gene was also determined. In both the imipramine- and fluoxetine-treated animals, expression of six genes was down-regulated (ANOVA-filtered gene expression data using dChip [version 2005]): Gpd1; Lrrn3; Sult1A1; Angptl4; Mt1a; Unknown. Furthermore, four genes were over-expressed: P4Ha1; RDG1311476; Rgc32; and SLC25A18-like by both imipramine and fluoxetine. These data demonstrate that antidepressant drugs interfere with the expression of genes involved in cell signaling, survival, and protein metabolism. Our results show that antidepressants regulate the induction of highly specific transcriptional programs in the developing frontal cortex. These findings provide novel insights into the long-term molecular actions of antidepressant drugs in the developing brain.

Cell persistence and electrical integration of transplanted fetal cardiomyocytes from different developmental stages

Cell replacement therapy is a promising approach to overcome thelimited regenerative potential of damaged myocardium. Persistenceand functional integration of transplanted cardiomyocytes are crucialfor a safe and efficient cell replacement therapy. The mechanismspromoting cell persistence and integration are poorly understood. Thedevelopmental stage of transplanted cells appears to be an importantfactor, since adult cardiomyocytes do not survive transplantation [1],while immature cardiomyocytes do survive and integrate functionally[1,2]. During fetal development, cardiomyocytes undergo substantialstructural and electrophysiological alterations [3–5], but it has notbeen studied yet, whether different stages of fetal developmentinfluence persistence and functional integration of transplantedcardiomyocytes, and if there is a distinct immature developmentalstagethatpromotesanoptimalpersistenceandfunctionoftransplantedcardiomyocytes.Therefore,weinvestigatedcellpersistenceandelectro-physiological integration of transplanted murine fetal cardiomyocytes(FCM) from different developmental stages.FetalventriclesfromtransgenicCrl:OF1miceexpressingeGFPundercontrol of the alpha-actin promoter were harvested at days 9.5(earlyFCM),14.5(interFCM)and18.5(lateFCM)p.c.Injectionsofdisso-ciated FCM were performed at two different sites of the left ventricularwall (2 × 450.000 cells) in adult Crl:OF1 mice. Six days after surgery,viable slices of recipient hearts (150 μm thickness) were producedwith a microtome (Leica, Germany). Intracellular action potentialswererecordedwithglassmicroelectrodes(WPI,USA).Sliceswerestim-ulated focally in host tissue. Cell preparation, surgery and microelec-trode recordings were performed as described before [2].Cellpersistence was quantified using a score ranging from “0” to “3”(0 = no graft cells visible; 1 = few and small clusters of transplantedcells (Fig. 1A + C); 2 = larger clusters of transplanted cells; 3 = largeareas of transplanted cells (Fig. 1B)). Quantitative analysis of cell persis-tence by quantitative TaqMan real-time PCR (qPCR) was performed sim-ilarlyasdescribedbefore [6,7].Sliceswerelysated,andgenomicDNAwasprepared, which was then used for qP CR with primers against the genesfor eGFP (grafted cells) and for beta-actin (total cell count). Data weretested for statistical significance by one-way ANOVA with post test or, ifnormality test failed, with Kruskal–Wallis test. Data are presentedas mean ± SEM. All experiments were approved by the local animalwelfare committee, and all animals received humane care.Persistence score was 2.91 ± 0.09 (n = 11) in interFCM, whichwas significantly higher compared to lateFCM (1.04 ± 0.12, n = 23,p b 0.001)and earlyFCM(0.40 ± 0.11,n = 20, p b 0.001vs.interFCM,p b 0.05 vs. lateFCM, Fig. 1D). The qPCR analysis confirmed thesefindings with 15,189 ± 4791 transgenic cells per mg detected inrecipient heart slices after injection of interFCM (n = 11), 3972 ±1730 cells/mg for lateFCM (n = 20, p b 0.05 vs. interFCM) and2290 ± 1851 cells/mg for earlyFCM (n = 17, p b 0.01 vs. interFCM,p = n.s. vs. lateFCM; Fig. 1E). Persistence of earlyFCM was insufficientfor electrophysiological analyses.The maximal stimulation frequency without conduction blocks,which was used to indicate the quality of integration, was8.62 ± 0.42 Hz in interFCM (n = 13) and 4.60 ± 0.67 Hz in lateFCM(n = 10, p b 0.001, Fig. 2A).Host cellsfollowed higherstimulation fre-quencies(11.00 ± 0.30 Hz;n = 21).Toquantifythevelocityofexcita-tion spread, we analyzed the delay between stimulus and actionpotential upstroke in host and graft cardiomyocytes. This delay waslower in interFCM (15.14 ± 1.03 ms, n = 18) than in lateFCM(28.49 ± 1.98 ms,n = 16,p b 0.001, Fig.2B + D),butwassignificant-ly higher in all transplanted FCM than in host cardiomyocytes(7.71 ± 0.21 ms, n = 28, pb 0.001 vs. interFCM and lateFCM). Thedistance between stimulation and recording electrode was equal inthese measurements (stimulation electrode to host cardiomyocytes1.51 ± 0.09 mm, to transplanted interFCM 1.55 ± 0.02 mm, totransplanted lateFCM 1.29 ± 0.16 mm; p = n.s.). Action potential

Developmental Regulation of Diacylglycerol Acyltransferase Family Gene Expression in Tung Tree Tissues

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.

The Prognostic Value of MicroRNAs Varies with Patient Race/Ethnicity and Stage of Colorectal Cancer

MicroRNAs (miRNA) have potential prognostic value for colorectal cancers; however, their value based on patient race/ethnicity and pathologic stage has not been determined. The goal was to ascertain the prognostic value of 5 miRNAs with increased expression in colorectal cancers of African American (black) and non-Hispanic Caucasian (white) patients. TaqMan quantitative real-time PCR was used to quantify expression of miR-20a, miR-21, miR-106a, miR-181b, and miR-203 in paired normal and tumor colorectal cancer archival tissues collected from 106 black and 239 white patients. The results were correlated with overall survival based on patient race/ethnicity and pathologic stage. Because decisions about adjuvant therapy are important for stage III colorectal cancers, and because miR-181b seemed to have prognostic value only for stage III black patients, we assessed its prognostic value in a separate cohort of 36 stage III colorectal cancers of blacks. All 5 miRNAs had higher expression in colorectal cancers (>1.0-fold) than in corresponding normal tissues. High expression of miR-203 was associated with poor survival of whites with stage IV colorectal cancers (HR = 3.00; 95% CI, 1.29-7.53), but in blacks it was an indicator of poor survival of patients with stages I and II colorectal cancers (HR = 5.63; 95% CI, 1.03-30.64). Increased miR-21 expression correlated with poor prognosis for white stage IV patients (HR = 2.50; 95% CI, 1.07-5.83). In both test and validation cohorts, high miR-181b expression correlated with poor survival of only black patients with stage III colorectal cancers (HR = 1.94; 95% CI, 1.03-3.67). These preliminary findings suggest that the prognostic value of miRNAs in colorectal cancers varies with patient race/ethnicity and stage of disease.

TaqMan real time-quantitative PCR targeting the phosphotransacetylase gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese

Late blowing defect affects hard cheese production and Clostridium tyrobutyricum appears as the primary cause of this spoilage. Our aim was to develop a quantitative TaqMan real-time PCR method to detect this agent in different matrices along the whole agro-dairy chain, using specific primers targeting the phosphotransacetylase gene (pta). The optimised assay appeared to be sensitive and specific to quantify C. tyrobutyricum. Results demonstrated a 100% efficiency with low numbers of cells and spores (approx. 14 mL−1 or g−1) also detected in broth and in milk matrix artificially contaminated with C. tyrobutyricum. Combined with a DNA extraction method optimised to lyse both cells and spores, our method is suitable for silage and hay, faeces, milk and hard cheese samples without any enrichment. The molecular quantification by qPCR was found to be precise and could be used for the rapid determination of the presence of C. tyrobutyricum.

Frequency and Expression of Inhibitory Markers of CD4+CD25+FOXP3+ Regulatory T Cells in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is the most symptomatic primary antibody deficiency associated with recurrent infections and chronic inflammatory diseases as well as autoimmunity. CD4(+) CD25(+) FOXP3(+) regulatory T cells (Tregs) are critical T cell subsets for maintaining self-tolerance and regulation of immune response to antigens thus play a pivotal role in preventing autoimmunity. Thirty-seven CVID patients and 18 age-/sex-matched controls were enrolled. Peripheral blood mononuclear cells (PBMCs) were obtained from both groups, and the percentage of Tregs was calculated using flow cytometry method. The mRNA expression of Tregs' surface markers cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor receptor (GITR), which are associated with Tregs' inhibitory function, was compared between patients and controls by quantitative real-time PCR TaqMan method. The results revealed that the frequency of Tregs was significantly lower in CVID patients than normal individuals (P<0.001). In addition, CVID patients with autoimmunity were found to have markedly reduced proportion of Tregs compared to those cases without autoimmune diseases (P=0.023). A significant difference was seen in factor forkhead box P3 (FOXP3) expression between CVID patients and controls (P<0.001). The mRNAs of CTLA-4 and GITR genes were expressed at lower levels in CVID patients compared to control group (P=0.005 and <0.001, respectively). Our findings showed reduced proportion of Tregs in CVID patients together with downregulation of FOXP3 protein and diminished expression of inhibitory Tregs' markers. It could be concluded that all of these changes may be responsible for cellular immune dysregulation observed in these patients especially those with autoimmune manifestation.

Abstract 4161: Genistein inhibits prostate cancer through activation of tumor suppressor microRNA-574-3p.

Abstract Introduction: Genistein has been shown to inhibit various cancers but its mechanism of action is still unknown. In this study, we tested the hypothesis that genistein can inhibit prostate cancer (PCa) through modulation of microRNA (miRNAs) using various in vitro models. We also tested the functional significance of miRNAs in PCa cell lines and clinical significance of miRNAs in different stages and grades of PCa. Methods: Subconfluent cells (60-70% confluent) were treated with genistein (25 μM). Genistein administered everyday along with a change of medium, and the cells were grown for 96 h. The miRNA microarray analysis was carried out and analyzed by Phalanx Biotech using the human v3 miRNA OneArray platform that is designed to contain 100% of miRBase Sequence Database Release 17.0. The expression levels of miR-574-3p were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. The 48 clinical specimens were obtained from patients in San Francisco Veterans Affairs Medical Center from 1998 to 2004. For gain-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), invasion (matrigel invasion assay) and apoptosis (flow cytometry analysis) were evaluated in human PCa cell lines, PC3 and DU145. Predicted target genes and their target miRNA binding site seed regions were investigated using TargetScan. To identify the biological processes or pathways potentially regulated by miR-574-3p, we performed GeneCodis analysis. To identify networks among the miRNAs and their target genes, we analyzed and characterized those genes in KEGG pathway categories. Results: Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3’ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Conclusions: Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa. Citation Format: Takeshi Chiyomaru, Soichiro Yamamura, Shinichiro Fukuhara, Hideo Hidaka, Shahana Majid, Sharanjot Saini, Sumit Arora, Guoren Deng, Varahram Shahryari, Inik Chang, Yuichiro Tanaka, Z. Laura Tabatabai, Hideki Enokida, Naohiko Seki, Masayuki Nakagawa, Rajvir Dahiya. Genistein inhibits prostate cancer through activation of tumor suppressor microRNA-574-3p. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4161. doi:10.1158/1538-7445.AM2013-4161

Abstract 4180: miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration.

Abstract Background: Glioblastoma is an aggressive tumor which originates from the glial cells and accounts for nearly 50% of adult brain tumors. These tumors are characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. MicroRNAs (miRNAs) are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that is used for the treatment of cardiovascular disease and various metabolic disorders. In addition, treatment with Fenofibrate results in the proliferation of peroxisomes and a marked reduction in the growth of Glioblastoma cells. Methods: To evaluate the effects of treatment on miRNA expression, we used TaqMan quantitative real-time PCR assays from Life Technologies (Grand Island, NY). miRNAs were cloned into BLOCK-iT Pol II miR RNAi Expression Vectors (Life Technologies). Cell growth was evaluated using Guava ViaCount Reagent (Guava Technologies, Hayward, CA). Migration of miRNA-expressing cells was assessed 24h after plating on 8 μm membranes (BD Biosciences, San Jose, CA). Results: In this study, we utilized the LN229 Glioblastoma cell line and found that among the miRNAs up-regulated by Fenofibrate treatment, miR-634 and miR-136 targeted CYR61, a protein involved in migration and proliferation of cancer cells. We additionally found that miR-136 was up-regulated by Fenofibrate treatment while miR-155, another miRNA previously shown to target CYR61, was down-regulated by Fenofibrate. All three miRNAs were predicted and validated to directly target the 3’-UTR of Cyr61. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, of LN229 cells. Forced expression of miR-155 reduced migration and addition of recombinant Cyr61 reversed this impairment in migration. Investigation of the signaling pathways regulated by miR-634, revealed an increased phosphorylation of p70 S6 kinase (Thr389), suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Induction of the mTOR pathway was dependent on Akt and Erk activation. In miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Further studies on the differential roles of miRs-136, -155, and -634 in controlling cell growth, cell size, and migration will provide important information on the molecular mechanisms involved in the anticancer properties of Fenofibrate. Citation Format: Duane Jeansonne, Marco Pacifici, Adam Lassak, Krzysztof Reiss, Francesca Peruzzi. miRNA-mediated anticancer properties of the PPARα-activator fenofibrate on glioblastoma cell growth and migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2013-4180

327 GENISTEIN INHIBITS PROSTATE CANCER THROUGH ACTIVATION OF TUMOR SUPPRESSOR MICRORNA-574-3P

327 GENISTEIN INHIBITS PROSTATE CANCER THROUGH ACTIVATION OF TUMOR SUPPRESSOR MICRORNA-574-3P

Development of a versatile TaqMan™ real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA

BackgroundIn bacterial systems, the sequence congruence of genomic DNA (gDNA) and cDNA obtained following reverse transcription of RNA, makes gDNA an automatic target for qPCR primers. This could lead to aberrant gene expression quantification. This is why a rigorous treatment of bacterial RNA with DNase I is usually required to remove any traces of carryover gDNA. As bacterial RNA is known to be extremely labile, any procedure that affects RNA yield, such as DNase I treatment, can be logically assumed to also influence detection and quantification of gene transcripts, leading to either an underestimation or no detection at all. To address such problems, we have developed a novel and versatile TaqMan RT-qPCR compliant anchor sequence (MYT4) for quantifying bacterial gene transcripts without the need for DNase I treatment.ResultsA non-genomic anchor sequence, henceforth referred to as MYT4 was designed using a synthetic DNA sequence called myIC, previously shown to share no significant homology to any known accession in the GenBank database. The sequence characteristic of MYT4 was kept within the design parameters required for the TaqMan RT-qPCR platform. The specificity and robustness of the novel MYT4 sequence was validated on RNA extracted from the bacterium Pseudomonas sp. LBUM300, grown under liquid culture and spiked soil conditions. Two transcripts, namely hcnC and phlD, were quantified from these two experimental systems. Using the MYT4 anchor, no RT-qPCR signal was detected from non-DNase I treated RNA, while strong signals were obtained using conventional reverse primers and RT-qPCR, indicating the presence of carryover gDNA in the RNA, extracted from either liquid culture or soil. Serial treatment of the RNA samples with DNase I (required to achieve absolute gDNA elimination) resulted in 50-70% loss of RNA which, when submitted to conventional RT-qPCR, significantly altered the transcript numbers detected when compared to the MYT4-based approach.ConclusionsImplementation of the versatile approach described in this study, which can be “retrofitted” to any existing TaqMan RT-qPCR system, should contribute to reducing the time and lowering the costs required to perform adequate bacterial RNA purification for downstream quantification of gene transcripts.

Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species

Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species. Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted Cryptosporidium andersoni and Cryptosporidium bovis as well as both human-pathogenic Cryptosporidium parvum and Cryptosporidium hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible, and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in the presence of non-target DNA in environmental samples. All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. No molecular-based method was yet available for the quantitative detection of C. andersoni and the cluster formed by C. bovis, Cryptosporidium ryanae and Cryptosporidium xiaoi. This work provides a novel tool to evaluate the parasite load from domestic ruminants and humans, and to improve assessment and management of microbial risk through better appraisal of the origin and fate of faecal pollutions.

Effect of alcohol exposure on hepatic superoxide generation and hepcidin expression

To understand the role of mitochondrial-produced superoxide (O2 (•-)) in the regulation of iron-regulatory hormone, hepcidin by alcohol in the liver. For alcohol experiments, manganese superoxide dismutase knockout mice heterozygous for Sod2 gene expression (Sod2 (+/-)) and age-matched littermate control mice (LMC), expressing Sod2 gene on both alleles, were exposed to either 10% (w/v) ethanol in the drinking water or plain water (control) for 7 d. Total cellular O2 (•-) levels in hepatocytes isolated from the livers of mice were measured by electron paramagnetic resonance spectroscopy. The mitochondrial-targeted, O2 (•-)-sensitive fluorogenic probe, MitoSOX Red and flow cytometry were utilized to measure O2 (•-) in mitochondria. Gene and protein expression were determined by Taqman Real-time quantitative PCR and Western blotting, respectively. Sod2 (+/-) mice expressed 40% less MnSOD protein (SOD2) in hepatocytes compared to LMC mice. The deletion of Sod2 allele did not alter the basal expression level of hepcidin in the liver. 10% ethanol exposure for 1 wk inhibited hepatic hepcidin mRNA expression three-fold both in Sod2 (+/-) and LMC mice. O2 (•-) levels in hepatocytes of untreated Sod2 (+/-) mice were three-fold higher than in untreated LMC mice, as observed by electron paramagnetic resonance spectroscopy. O2 (•-) levels in mitochondria of Sod2 (+/) mice were four-fold higher than in mitochondria of untreated LMC mice, as measured by MitoSOX Red fluorescence and flow cytometry. Alcohol induced a two-fold higher increase in O2 (•-) levels in hepatocytes of LMC mice than in Sod2 (+/-) mice compared to respective untreated counterparts. In contrast, 1 wk alcohol exposure did not alter mitochondrial O2 (•-) levels in both Sod2 (+/-) and control mice. Mitochondrial O2 (•-) is not involved in the inhibition of liver hepcidin transcription and thereby regulation of iron metabolism by alcohol. These findings also suggest that short-term alcohol consumption significantly elevates O2 (•-) levels in hepatocytes, which appears not to originate from mitochondria.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y=−3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =−3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1–4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.

Abstract 2299: Genistein suppresses prostate cancer growth through inhibition of oncogenic miR-151

Abstract Introduction and Objective: Genistein has been shown to suppress the growth of several cancer cell lines in vitro and in vivo, reducing the expression of several oncogenic microRNAs. However, the effects of genistein on the regulation of oncogenic miR-151 have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer. Methods: The expression levels of miR-151 were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. Nineteen clinical specimens were obtained from patients at the San Francisco Veterans Affairs Medical Center from 2000 to 2006. For gain-and loss-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), and invasion (matrigel invasion assay) were evaluated in human prostate cancer cell lines, PC3 and DU145. Predicted target genes and their target microRNA binding site seed regions were investigated using TargetScan and microRNA.org. Genistein was administered every day along with a change of medium, and the cells were grown for four days. Results: Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 μM genistein down-regulated the expression of miR-151 compared with vehicle control. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had a significant adverse effect on survival rate. Inhibition of miR-151 in prostate cancer cells by genistein significantly inhibited cell migration and invasion. By in-cilico analysis, several genes (N4BP1, CASZ1, IL1RAPL1, LGI1, SOX17 and TOM1L1) that are suggested to have tumor suppressive function in several types of cancers were detected as candidate target genes of miR-151. Conclusions: We demonstrated that genistein down-regulated miR-151 that functions as an oncogenic miRNA in prostate cancer. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of prostate cancer. Source of Funding: Grants R01CA160079-01, R01CA138642, VA Research Enhancement Award Program (REAP), and VA Merit Review grants Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2299. doi:1538-7445.AM2012-2299

Abstract 117: The processed pseudogene POU5F1P1in 8q24 is expressed in tumor and shows oncogenicity

Abstract Genome-wide association studies (GWAS) have identified a segment of approximately 1 Mb of 8q24 as a multi-cancer cancer susceptibility region. The region is flanked by two genes, FAM84B and MYC, the latter an established oncogene. Within this region of 8q24, there is a processed pseudogene, POU5F1P1. We have re-sequenced the exon regions of wild type POU5F1 on chromosome 6 and its six counterpart pseudogenes on chromosomes 1, 3, 8, 10 and 12, and identified sequence variants unique to POU5F1P1 for which we have developed and confirmed analysis of expression of the POU5F1P1 transcript on 8q24. The homolog between wild type POU5F1 and pseudogene POU5F1P1 is about 97%. By using TaqMan DNA copy number variation assays, we identified uncommon copy number variants across this region. However, the DNA copy number variation does not contribute to differences in expression of POU5F1P1. Expression of POU5F1P1 was confirmed in a subset of the NCI60 panel using quantitative TaqMan real-time PCR assays in cell lines of colon, ovarian and breast cancers. Both POU5F1 and POU5F1P1were further evaluated in 14 colon cancer cell lines of which two, DLD-1 and KM12, expressed POU5F1P1 but no wild type POU5F1. Using shRNA technology (two constructs for each target), we knocked down POU5F1P1 expression in DLD-1 and KM12, which lead to reduction in the growth rate and anchorage-independent growth of colon cancer cells. Our results suggest that the processed pseudogene, POU5F1P1 is expressed in cell lines and colon cancer tissues. Moreover, preliminary studies suggest that its function could be related to oncogenic activity, though further studies are needed to verify this observation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 117. doi:1538-7445.AM2012-117

Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs.

Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\\103–6 times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogenThe finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.

MicroRNA-15a/b are up-regulated in response to myocardial ischemia/reperfusion injury

ObjectiveSeveral studies have indicated that miR-15a, miR-15b and miR-16 may be the important regulators of apoptosis. Since attenuate apoptosis could protect myocardium and reduce infarction size, the present study was aimed to find out whether these miRNAs participate in regulating myocardial ischemia reperfusion (I/R) injury.MethodsApoptosis in mice hearts subjected to I/R was detected by TUNEL assay in vivo, while flow cytometry analysis followed by Annexin V/PI double stain in vitro was used to detect apoptosis in cultured cardiomyocytes which were subjected to hypoxia/reoxygenation (H/R). Taqman real-time quantitative PCR was used to confirm whether miR-15a/15b/16 were involved in the regulation of cardiac I/R and H/R.ResultsCompared to those of the controls, I/R or H/R induced apoptosis of cardiomyocytes was significantly increased both in vivo (24.4% ± 9.4% vs. 2.2% ± 1.9%, P < 0.01, n = 5) and in vitro (14.12% ± 0.92% vs. 2.22% ± 0.08%). The expression of miR-15a and miR-15b, but not miR-16, was increased in the mice I/R model, and the results were consistent in the H/R model.ConclusionsOur data indicate miR-15 and miR-15b are up-regulated in response to cardiac I/R injury, therefore, down-regulation of miR-15a/b may be a promising strategy to reduce myocardial apoptosis induced by cardiac I/R injury.

Strain‐specific modifier genes governing craniofacial phenotypes

The presence of strain-specific modifier genes is known to modulate the phenotype and pathophysiology of mice harboring genetically engineered mutations. Thus, identification of genetic modifier genes is requisite to understanding control of phenotypic expression. c-Ski is a transcriptional regulator. Ski(-/-) mice on a C57BL6J (B6) background exhibit facial clefting, while Ski(-/-) mice on a 129P3 (129) background present with exencephaly. In the present study, oligonucleotide-based gene expression profiling was used to identify potential strain-specific modifier gene candidates present in wild type mice of B6 and 129 genetic backgrounds. Changes in gene expression were verified by TaqMan quantitative real-time PCR. Steady-state levels of 89 genes demonstrated a significantly higher level of expression, and those of 68 genes demonstrated a significantly lower level of expression in the developing neural tubes from embryonic day (E) 8.5, B6 embryos when compared to expression levels in neural tubes derived from E 8.5, 129 embryos. Based on the results from the current comparative microarray study, and taking into consideration a number of relevant published reports, several potential strain-specific gene candidates, likely to modify the craniofacial phenotypes in various knockout mouse models have been identified.