Development and Validation of Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction for Monitoring Gene Expression in Cardiac Myocytesin Vitro
ABSTRACT In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.
- SourceAvailable from: Zhangang Xiao[Show abstract] [Hide abstract]
ABSTRACT: Prothymosin alpha (ProTα) and thymosin beta (Tβ) belong to thymosin family, which consists of a series of highly conserved peptides involved in stimulating immune responses. ProTα b and Tβ are still poorly studied in teleost. Here, the full-length cDNAs of ProTα b and Tβ-like (Tβ-l) were cloned and identified in common carp (Cyprinus carpio L.). The expressions of carp ProTα b and Tβ-l exhibited rise-fall pattern and then trended to be stable during early development. After spring viraemia of carp virus (SVCV) infection, the carp ProTα b and Tβ-l transcripts were significantly up-regulated in some immune-related organs. When transiently over-expressed carp ProTα b and Tβ-l in zebrafish, these two proteins up-regulated the expressions of T lymphocytes-related genes (Rag 1, TCR-γ, CD4 and CD8α). These results suggest that carp ProTα b and Tβ may ultimately enhance the immune response during viral infection and modulate the development of T lymphocytes in teleost. Copyright © 2015. Published by Elsevier Ltd.Developmental & Comparative Immunology 01/2015; 50(1). DOI:10.1016/j.dci.2015.01.003 · 3.71 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Arbuscular mycorrhizal fungi (AMF) are known to improve plants stress tolerance by increasing water absorption and trehalose accumulation. However, the relationship between trehalose and AMF water transport and their role in the mycorrhizal rice water strategy at low temperature has not been established. Different temperature and exogenous trehalose treatment experiments were performed on potted non-mycorrhizal and mycorrhizal rice. The results showed that AMF enhanced rice root water uptake at both normal and low temperatures. At low temperature, the AMF aquaporin (GintAQPF) expression levels were increased and higher rice aquaporin (OsPIPs) expression levels were exhibited in mcyorrhizal rice than non-mycorrhizal rice. The increased trehalose biosynthesis gene transcripts, such as OsTPS1, OsTPS2 and OsTPP1 in mycorrhizal rice roots generated more trehalose than in non-mycorrhizal rice at either normal or low temperature. Application of exogenous trehalose demonstrated that trehalose could regulate AMF and rice water absorption by inducing the expression of GintAQPF and several OsPIPs and create better plant growth conditions. Consequently, we hypothesized that one of the mechanisms by which AMF improve plant resistance to low temperature was AMF-enhanced trehalose accumulation, which could induce AMF and host plants aquaporin expression that then maintain better water relations in mycorrhizal plants at low temperatures.Applied Soil Ecology 12/2014; 84:185–191. DOI:10.1016/j.apsoil.2014.07.010 · 2.21 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The fruit of Ziziphus jujuba Mill., known as jujube or Chinese date, is commonly consumed as a health supplement or herbal medicine worldwide. To study the beneficial role of jujube in regulating immune response, we investigated its roles on the expressions of pro-inflammatory cytokines in cultured macrophages. Application of chemically standardized jujube water extract for 24 h stimulated the transcriptional expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in cultured RAW 264.7 macrophages. In contrast, the pretreatment with jujube water extract suppressed the expression of IL-1β and IL-6, but not for TNF-α in lipopolysaccharide (LPS)-stimulated macrophages. The IL-1β and IL-6 cytokines in LPS-induced macrophages were suppressed by jujube water extract in both mRNA and protein levels. In parallel, the inhibition of jujube water extract on the transcriptional activity of nuclear factor-kappa B was revealed in LPS-induced macrophages. These results verified the bidirectional immune-modulatory roles of jujube by regulating the expressions of pro-inflammatory cytokines in macrophages. Copyright © 2014 John Wiley & Sons, Ltd.Phytotherapy Research 10/2014; 28(10). DOI:10.1002/ptr.5160 · 2.40 Impact Factor