Single-tube Multiplex Nested PCR System for Efficient Detection of Pathogenic Microorganisms in SPF Rodents.

PCR testing is increasingly important for microbial control in SPF facilities. However, most current PCR methods are timeconsuming and require compromise between high sensitivity and high multiplexing. We developed a one-tube multiplex nested PCR strategy (MN-PCR) for simultaneous direct (that is, without culturing) detection of multiple pathogens.

The role of Dby mRNA in early development of male mouse zygotes.

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected.

Nicotine induces cyclooxygenase-2 and prostaglandin E(2) expression in human umbilical vein endothelial cells.

Nicotine is a major component of cigarette smoking which may be involved in the progress of atherogenesis. In order to explain the mechanism of nicotine-induced endothelium dysfunction, we investigated the effects of nicotine on cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVECs). Nicotine treatment increased the expressions of COX-2 at mRNA and protein level in a dose-dependent manner, following prostaglandin E(2) (PGE(2)) release enhancement.

Modified PCR methods for 3' end amplification from serial analysis of gene expression (SAGE) tags.

Serial analysis of gene expression (SAGE) is a powerful technique to study gene expression at the genome level. However, a disadvantage of the shortness of SAGE tags is that it prevents further study of SAGE library data, thus limiting extensive application of the SAGE method in gene expression studies. However, this problem can be solved by extension of the SAGE tags to 3' cDNAs.

Semi-nested PCR analysis of unknown tags on serial analysis of gene expression.

Serial analysis of gene expression (SAGE) is a powerful technique for studying gene expression at the genome level. However, short SAGE tags limit the further study of related data. In this study, in order to identify a gene, we developed a semi-nested PCR-based method called the two-step analysis of unknown SAGE tags (TSAT-PCR) to generate longer 3'-end cDNA fragments from unknown SAGE tags.

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a Trichoplusia ni cell line.

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed.