SYBR Green Quantitative RT-PCR Research Articles

Analogous telomeres shortening and different metabolic profile: hypertension versus hypertension/type 2 diabetes mellitus comorbidity.

Eukaryotes chromosomal ends are capped and protected by telomeres, which are noncoding DNA repeats synthesized by telomerase enzyme. The telomerase enzyme is a nucleoprotein encoded by TERC and TERT genes. Naturally, the length of the telomeres shortens with each cell cycle but the shortening is fastened in certain age-related diseases like hypertension (HTN) and type 2 diabetes mellitus (T2DM). Blood samples (n = 171) were obtained from Kuwaiti subjects with HTN, and HTN/T2DM comorbidity (HTN-DM) and healthy subjects. The leukocyte telomere length (LTL) was measured by SYBR green quantitative rtPCR, and plasma telomerase enzyme was measured by ELISA, in addition, three single nucleotide polymorphisms (SNPs) in telomere-related genes; TERC rs12696304GC, TERT rs2736100CA, and ACYP2 rs6713088GC were genotyped by real-time PCR. Marked LTL shortening in subjects with HTN and HTN-DM compared to healthy subjects, P = 0.043 and P < 0.001, respectively, was noticed. On the contrary, the plasma telomerase enzyme levels and minor allele frequencies and genotypes of the tested SNPs were comparable between the study groups, except for TERT (CA) genotype which was over-represented in HTN (P = 0.037). Furthermore, the comparisons between HTN and HTN-DM revealed significantly higher total cholesterol (P = 0.015) and LDL-C (P = 0.008) in HTN, while higher insulin levels (P < 001), HOMA-IR (P < 001), and BMI (P = 0.004) were observed in HTN-DM. This study showed comparable LTL shortening in HTN and HTN-DM, irrespective of plasma telomerase enzyme levels or tested TERC, TERT, and ACYP2 gene polymorphisms, although HTN and HTN-DM differed in several metabolic markers. More studies are required to affirm these observations.

Diagnostic and prognostic roles of circulating miRNA-223-3p in hepatitis B virus-related hepatocellular carcinoma.

Circulating microRNAs (miRNAs) have been shown to dysregulate in many cancer types including hepatocellular carcinoma (HCC). The purpose of this study was to examine the potential diagnostic or prognostic roles of circulating miRNAs in patients with hepatitis B virus (HBV)-related HCC. Paired cancerous and adjacent non-cancerous liver tissue specimens of patients with HBV-related HCC were used as a discovery set for screening 800 miRNAs by a Nanostring quantitative assay. Differentially expressed miRNAs were then examined by SYBR green quantitative RT-PCR in a validation cohort of serum samples obtained from 70 patients with HBV-related HCC, 70 HBV patients without HCC and 50 healthy controls. The discovery set identified miR-223-3p, miR-199a-5p and miR-451a significantly lower expressed in cancerous tissues compared with non-cancerous tissues. In the validated cohort, circulating miR-223-3p levels were significantly lower in the HCC group compared with the other groups. The combined use of serum alpha-fetoprotein and miR-223-3p displayed high sensitivity for detecting early HCC (85%) and intermediate/advanced stage HCC (100%). Additionally, serum miR-223-3p had a negative correlation with tumor size and BCLC stage. On multivariate analysis, serum miR-223-3p was identified as an independent prognostic factor of overall survival in patients with HCC. In contrast, circulating miRNA-199a-5p and miR-451a did not show any clinical benefit for the diagnosis and prognostic prediction of HCC. Our results demonstrated that miR-223-3p was differentially expressed in cancerous compared with paired adjacent non-cancerous tissues. In addition, circulating miRNA-223-3p could represent a novel diagnostic and prognostic marker for patients with HBV-related HCC.

Differential HSP70 expression in Mytilus coruscus under various stressors

Heat shock proteins (HSPs) play crucial roles in protecting cells against environmental stresses, such as heat shock, heavy metals and pathogenic bacteria. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. The full-length HSP70 cDNA of Mytilus coruscus (designated as McHSP70, GenBank accession no. KF322135) was obtained, which was 2319 bp, including an ORF of 1965 bp to encode a polypeptide of 655 amino acid with predicted pI/MW5.29/71.38 kDa, a 81 bp-5'-UTR and a 270 bp-3'-UTR. The BLAST analysis and phylogenetic relationship strongly suggested that this amino acid sequence was a member of HSP70 family and highly homologous with Mytilus galloprovincialis (95%). Multiple sequence alignment revealed that McHSP70 and other known HSP70 were highly conserved, especially in the regions of HSP70 family signatures, the bipartite nuclear targeting sequence, ATP/GTP-Binding site motif and 'EEVD' motif. The mRNA of McHSP70 in hemolymph was constitutively expressed in all treatments including Vibrio challenge, thermal stress, metals (copper and cadmium) and 180 CST fuel exposure based on SYBR Green quantitative RT-PCR analysis. The temporal expression of HSP70 mRNA in hemolymph was significantly affected by Vibrio alginolyticus and Vibrio harveyi challenge. The maximum level appeared at 24h post-injection with 5.44-fold in V. alginolyticus and dropped back to the original level at 72 h post-injection. In V. harveyi infection the transcripts of McHSP70 was significantly induced to the highest at 2h after post-injection with 13.52-fold, then decreased until 36 h appearing another expression peak with 10.29-fold, and returned gradually to physiological level at the end of this experiment. In heat shock experiment the maximum expression appeared at 12h with 15-fold higher than that of blank mussels. Time-dependent mRNA expression pattern of McHSP70 was found in exposure to copper, cadmium and 180 CST fuel, but the highest expression and time were different in various treatments (copper of 9.41-fold at day 15, cadmium of 10.82-fold at day 10, and 180 CST fuel of 5.94-fold at day 25), and then dropped progressively. All these results indicated that HSP70 in marine bivalve had a significant role in mediating the environmental stress and immune response.

Signalling molecules involved in mouse bladder smooth muscle cellular differentiation

Mouse bladder mesenchyme differentiates into smooth muscle under the influence of urothelium at gestational day 13.5 (E13.5). Sonic hedgehog (Shh) is considered to be the upstream gene arising from the urothelium, which induces smooth muscle in the peripheral bladder mesenchyme. We hypothesize differential gene expression across the full thickness of bladder mesenchyme as a function of proximity to the inducing bladder urothelium and the peripheral location of the smooth muscle. Embryonic bladders from FVB mice were collected at E12.5, 13.5, 15 and 16 and cryosectioned followed by microdissection with a PixCell II laser capture microscope. RNA extraction was performed at the laser captured sites and mRNA expression profiles were measured using SYBR Green quantitative RT-PCR. Smooth muscle a-actin (SMAA) and smooth muscle myosin heavy chain (SM-MHC) were expressed in the E13.5, E15 and E16 bladders in the peripheral layer of mesenchyme, but not in the prospective submucosa. Patched 1 (Ptc1), Gli1 and bone morphogenetic protein (Bmp) 4 expression was consistently elevated in the mesenchymal layer immediately adjacent to the urothelium compared to the peripheral location at E12.5. After E12.5, Ptc1 expression decreased to an undetectable level throughout the bladder mesenchyme. The level of TGF-beta1 was highest in the mesenchymal layer adjacent to the serosa at E13.5. The level of expression of serum response factor (SRF) was also highest at E15 in the peripheral mesenchyme. Genes downstream of Shh are differentially expressed in the prospective submucosa vs. the peripheral bladder mesenchyme as a function gestation age and smooth muscle differentiation.

Gene Microarray Analysis for Porcine Adipose Tissue: Comparison of Gene Expression between Chinese Xiang Pig and Large White

We created a cDNA microarray representing approximately 3,500 pig genes for functional genomic studies. The array elements were selected from 6,494 cDNA clones identified in a large-scale expressed sequence tag (EST) project. These cDNA clones came from normalized and subtracted porcine adipose tissue cDNA libraries. Sequence similarity searches of the 3,426 ESTs represented on the array using BLASTN identified 2,790 (81.4%) as putative human orthologs, with the remainder consisting of novel genes or highly divergent orthologs. We used the gene microarray to profile transcripts expressed by adipose tissue of fatty Chinese Xiang pig (XP) and muscley Large White (LW). Microarray analysis of RNA extracted from adipose tissue of fatty XP and muscley LW identified 81 genes that were differently expressed two fold or more. Transcriptional differences of four of these genes, adipocyte fatty acid binding protein (aP2), stearyl-CoA desaturase (SCD), sterol regulatory element binding transcription factor 1 (SREBF1) and lipoprotein lipase (LPL) were confirmed using SYBR Green quantitative RT-PCR technology. Our results showed that high expression of SCD and SREBF1 may be one of the reasons that larger fat deposits are observed in the XP. In addition, our findings also illustrate the potential power of microarrays for understanding the molecular mechanisms of porcine development, disease resistance, nutrition, fertility and production traits.

Chromatin Modification and the Endothelial-specific Activation of the E-selectin Gene

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) alpha induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-kappa B (NF-kappa B) to the endogenous E-selectin promoter after TNFalpha stimulation along with IkappaB kinase alpha. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFalpha induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells.

Neuroprotective Effects of Phenylbutyrate in the N171-82Q Transgenic Mouse Model of Huntington's Disease

Huntington's disease (HD) is caused by an expansion of exonic CAG triplet repeats in the gene encoding the huntingtin protein (Htt), however, the means by which neurodegeneration occurs remains obscure. There is evidence that mutant Htt interacts with transcription factors leading to reduced histone acetylation. We report that administration of the histone deacetylase inhibitor phenylbutyrate after onset of symptoms in a transgenic mouse model of HD significantly extends survival and attenuates both gross brain and neuronal atrophy. Administration of phenylbutyrate increased brain histone acetylation and decreased histone methylation levels as assessed by both immunocytochemistry and Western blots. Phenylbutyrate increased mRNA for components of the ubiquitin-proteosomal pathway and down-regulated caspases implicated in apoptotic cell death, and active caspase 3 immunoreactivity in the striatum. These results show that administration of phenylbutyrate, at doses that are well tolerated in man, exerts significant neuroprotective effects in a transgenic mouse model of HD, and therefore represents a very promising therapeutic approach for HD.