Relative Quantitative Real-time PCR Research Articles

QPCR-DAMS: a database tool to analyze, manage, and store both relative and absolute quantitative real-time PCR data

Quantitative real-time PCR is an important high-throughput method in the biomedical sciences. However, existing software has limitations in handling both relative and absolute quantification. We designed quantitative PCR data analysis and management system (qPCR-DAMS), a database tool based on Access 2003, to deal with such shortcomings by the addition of integrated mathematical procedures. qPCR-DAMS allows a user to choose among four methods for data processing within a single software package: 1) ratio relative quantification, 2) absolute level, 3) normalized absolute expression, and 4) ratio absolute quantification. qPCR-DAMS also provides a tool for multiple reference gene normalization. qPCR-DAMS has three quality control steps and a data display system to monitor data variation. In summary, qPCR-DAMS is a handy tool for real-time PCR users.

Bone Microenvironment-related Growth Factors, Zoledronic Acid and Dexamethasone Differentially Modulate PTHrP Expression in PC-3 Prostate Cancer Cells

Bone metastasis microenvironment-related growth factors such as insulin-like growth factor 1 (IGF-1), transforming growth factor beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF) and interleukin 6 (IL-6) show survival factor activity, thereby inhibiting chemotherapy-induced apoptosis of PC-3 prostate cancer cells in vitro. Recently, zoledronic acid has been shown to induce apoptosis in PC-3 prostate cancer cells while overexpression of parathyroid hormone-related protein (PTHrP) inhibits serum deprivation-induced apoptosis in PC-3 cells. Consequently, we have investigated whether IGF-1, TGF-beta1, bFGF, IL-6, zoledronic acid and/or dexamethasone affect the expression of the PTHrP and type I PTH/PTHrP receptor (PTH.1R) in PC-3 prostate cancer cells using relative quantitative PCR and real-time PCR (expression at mRNA level) and immunocytochemical and immunofluorescence analysis (expression at protein level). Our data show that IGF-1, TGF-beta1, bFGF and IL-6 increase PTHrP mRNA expression and its perinuclear localization, while zoledronic acid (50 muM, 100 muM for 24 h and 48 h) and dexamethasone suppress PTHrP expression in PC-3 cells. We did not detect any appreciable change of the PTH.1R expression due to IGF-1, TGF- beta1, bFGF, IL-6, zoledronic acid or dexamethasone in PC-3 cells. Therefore, it is conceivable that bone metastasis microenvironment-related survival factor/anti-apoptotic activity and zoledronic acid anticancer action/pro-apoptotic activity on PC-3 cells is mediated, at least in part, by differential modulation of PTHrP expression.

Immune challenge differentially affects transcript abundance of three antimicrobial peptides in hemocytes from the moth Pseudoplusia includens

Inducible expression of antimicrobial peptides and other humoral immune factors by the insect fat body is well documented. Hemocytes comprise the second essential arm of the insect immune system but it is unclear whether antimicrobial peptide genes are expressed by all or only some types of hemocytes. Here we report the cloning of cecropin A ( Pi-cecA), lebocin ( Pi-leb) and lysozyme ( Pi-lys) homologs from the moth Pseudoplusia includens. Relative-quantitative real-time PCR (rq-rtPCR) indicated that transcript abundance for each antimicrobial gene increased in fat body and hemocytes following immune challenge with the Gram-negative bacterium Escherichia coli. Relative transcript abundance of Pi-cecA was much higher in fat body than hemocytes. In contrast, transcript levels of Pi-leb were three-fold lower in hemocytes than fat body while transcript levels of Pi-lys were three-fold higher. Estimates for the overall contribution of the fat body and hemocytes to antimicrobial peptide expression suggested that hemocytes contribute significantly to Pi-lys transcript levels in larvae but produce much smaller amounts of Pi-cecA and Pi-leb compared to the fat body. Each antimicrobial peptide was also inducibly expressed in hemocytes following challenge with the Gram-positive bacterium Micrococcus luteus or when hemocytes formed capsules around chromatography beads. Analysis of hemocyte types indicated that granulocytes and plasmatocytes expressed all three antimicrobial peptides, whereas spherule cells and oenocytoids expressed only lysozyme. Transcriptional profiles of these antimicrobial genes were similar in granulocytes and plasmatocytes in vivo but were very different in vitro.

Cloning, developmental regulation and neural localization of rat ε-sarcoglycan

Mutations in the gene for epsilon sarcoglycan (ε-SG) are associated with a disorder of the central nervous system, the myoclonus-dystonia syndrome (MDS; DYT11). In contrast, mutations of other sarcoglycan family members lead to limb-girdle muscular dystrophies. To establish the framework for functional studies of ε-SG, we cloned rat ε-SG cDNA, quantified ε-SG mRNA levels in neural and non-neural tissues at different developmental time points with relative quantitative multiplex real-time reverse transcriptase PCR (RT-PCR), and characterized the distribution of ε-SG mRNA in brain with in situ hybridization. Rat ε-SG cDNA contains an open reading frame (ORF) of 1311 bp that encodes a 437-amino acid (aa) protein with 95.9% and 98.2% identity to human and mouse ε-SG amino acid sequences, respectively. Using real-time RT-PCR, ε-SG was detected in both neural (cerebellar cortex, striatum, cerebral cortex, thalamus, hippocampus) and non-neural (muscle, liver, kidney, heart) tissues at each developmental time point tested [Embryonic Day 20 (E20), Postnatal Day 1 (P1), P7, P14, P36, 6 months, 1.5 years). Levels of ε-SG mRNA were highest at E20 in all tissues. The developmental regulation of ε-SG mRNA expression was most striking in muscle with E20 and early postnatal ε-SG mRNA levels over 10 times higher than those seen in adult rats. In adult rats, ε-SG mRNA levels were several-fold higher in brain, particularly cerebellar cortex, than in muscle. Radioactive in situ hybridization showed that ε-SG mRNA was widely distributed in rat brain. Robust hybridization signal was obtained from regions with dense neuronal packing such as the hippocampus, cerebellar molecular layer, and cerebral cortex. Our results suggest that ε-SG participates in the development of both neural and non-neural tissues and contributes to neuronal structure in the adult central nervous system.