Pattern of Expression of γ‐Glutamyl Transpeptidase in Rat Liver and Kidney During Development
Emmanuel JacqueminFrédérique BulleJean‐François BernaudinMaria WellmanRose‐Noëlle HugonGeorges GuellaënMichelle Hadchouel
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Summary We used an in situ hybridization technique using single‐stranded RNA probes to study the expression pattern of γ‐glutamyl transpeptidase (GGT) in rat liver and kidney during development. The results were compared to those obtained with an immunoperoxidase technique and with Northern blot analysis of GGT mRNA. In the kidney, northern blot revealed a 20‐fold increase of GGT mRNA between day 18 of gestation and adulthood. Protein and mRNA localization clearly identified the proximal tubules as the site of synthesis of GGT. In the liver, the expression was lower than in the kidney and Northern blot showed a dramatic decrease of expression after birth. Using immunohistochemistry, the protein was detected within parenchymal cells in embryo and hepatocyte membranes and bile ducts in adults. Using in situ hybridization, GGT mRNA was only detected on days I and 2 after birth and exclusively in hepatocytes. Immunoperoxidase may be more sensitive than in situ hybridization to study the expression of minor liver protein such as GGT. However, the study of GGT expression using in situ hybridization is possible in cases of increased expression such as alcoholism, cholestasis, and carcinogenesis.Keywords:
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Orbivirus
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We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.
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SUMMARY Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope‐ and biotin‐ (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope‐based probe is limited by strict environmental regulation and availability in many places in the world while biotin‐based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase‐ b ased method for incorporation of a c luster of 33 biotin‐labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32 P‐labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low‐abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus‐based miRNA silencing in pepper, knocking down accumulation of Can‐miR6027a and Can‐miR6149L. Importantly, further analysis showed that knocking‐down Can‐miR6027a led to upregulation of a nucleotide binding‐leucine rich repeat domain protein coding gene ( CaRLb1 ) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici .
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Objective To estabilish dot blot hybridization technology in the detection of HIF-1a gene expression of various tumor cells and to explore the availability of dot blot in screening gene expression difference.Methods Nuclear acid were extracted from a variety of cultured tumor cell lines and spotted on the nylon membrane,dot blot analysis and semi-quantitative RT-PCR were carded out respectively to detect HIF-1a gene expression.Results The dot-blot hybridization results are consistent with the RT-PCR.it did not reach the conventional level of statistical significance(P0.05).Conclusion Dot blot hybridization with DIG-labeled HIF-1a probe can be used to screen quickly gene expressing difference in tumor cells and serve as a research tool for tumor gene expression difference.
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Objective
Use of biotin-labeled sRNA probes to detect Bacillus Calmette-Guerin (BCG) small RNA (sRNA).
Methods
The plasmid pMD18-T/ASdes was constructed according to sRNA-ASdes of BCG and used as a template to synthesize the ASdes (3′T7) bio-RNA probes with T7 RNA polymerase by in vitro transcription. Then, the sensibility of the probe was tested with Northern blot and Dot blot.
Results
The positive signal was obvious in Northern blot in detecting the sRNA-ASdes of BCG using ASdes (3′T7) bio-RNA probes.
Conclusion
The sensibility of the randomly biotin-labeled sRNA probes obtained by in vitro transcription was high, which could be widely used in the detection of sRNA in Northern blot.
Key words:
In vitro transcription; Randomly biotin-labeled; sRNA probe; Northern blot; Dot blot
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AIM To examine the expression levels of human er a mRNA in different tissues or tumor cell lines. METHODS Human era encoding region gene labeled with (dCTP was used as a probe and its mRNA levels were examined in multiple tissue northern (MTN) blot membrane and multiple tissue expression (MTE) array membrane by Northern blot and Dot blot h ybridization respectively. RESULTS Northern blot showed the pro be identified an mRNA product of approximately 2.2 kb, and Dot blot showed human era expressed in all tissues and cell lines, the highest expression found i n the nervous system, some fetal tissues and tumor cell lines. CONCLUSIO N Human era mRNA expresses in all tissues tested at different level s, so it may be a constitutively expressing gene.
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