A stable and differentiable RNA positive control for reverse transcription-polymerase chain reaction

Most RNA positive controls currently used for monitoring the quality of RT-PCR assays have some disadvantages, such as instability, inability to monitor the quality of the relevant primers and/or causing indifferentiable false positives. To avoid these disadvantages, a simple method to prepare stable and differentiable RNA positive controls is now demonstrated with a real-time RT-PCR assay for the detection of Nipah virus (NiV). A DNA sequence which was shorter than its counterpart in the NiV genome and contained the binding sites of the primers of the RT-PCR assay was designed, synthesized and inserted into a vector, and then amplified by PCR with two vector-specific primers both of which contained a T7 promoter at the 5' terminal. The RNA positive control was the dsRNA in vitro transcribed from the PCR amplicons flanked by two T7 promoters. The RNA positive control was stable and able to monitor the quality of the whole concerned RT-PCR assay. False positives caused by contaminations of the RNA positive control or its amplicons could be easily identified because the amplicons of the RNA positive control were obviously shorter than those of real positive samples. Thus, the RNA positive control reported in this study avoided some common disadvantages of current RNA positive controls.     

In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy

Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl₂, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.     

Quantitative analysis of estrogen receptor mRNA in human endometrium throughout the menstrual cycle using a real-time reverse transcription-polymerase chain reaction assay

We conducted a quantitative analysis of ERalpha and ERbeta mRNA expression in normal human endometrium throughout the menstrual cycle in regular menstruating premenopausal women, taking advantage of this real-time PCR assay. Endometrial dating was determined from the histology of the endometrium and classified into: proliferative endometrium and secretory endometrium. Both ERalpha and ERbeta mRNA expression were detected in all endometrial samples at both proliferative and secretion phase. However ERalpha mRNA expression level was higher than that of ERbeta specially during proliferative phase. These results suggest that estrogenic effects occur predominantly through ERalpha than ERbeta.