The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression pro®ling with single-cell resolution is highly desirable, in particular for complex tis...

Porcine respiratory disease complex (PRDC) continues to be a significant economic problem for swine producers. Porcine reproductive and respiratory syndrome virus (PRRSV), Swine influenza virus (SIV), Porcine circovirus-2 (PCV-2), Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, and Pasturella multocida are among the most important pathogens contributing to PRDC. Recently, a new gener...

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and Simplexa™ Dengue real-time RT-PCR confir...

Unlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the s...

We have developed a novel "real time" quantitative PCR method. The method measures PCR product accumulation through a dual-labeled fluorogenic probe (i.e., TaqMan Probe). This method provides very accurate and reproducible quantitation of gene copies. Unlike other quantitative PCR methods, real-time PCR does not require post-PCR sample handling, preventing potential PCR product carry-over conta...

Real-time reverse transcription polymerase chain reaction (RT-PCR) methods that monitor product accumulation were adapted for the validation of differentially expressed genes. We describe a real-time quantitative PCR assay that uses SYBR Green I dye-based detection and product melting curve analysis to validate differentially expressed genes identified by gene expression profiling technologies....

Those who have worked in the field of quantitative polymerase chain reaction (PCR) since the early 1990s have accepted many of the tedious aspects of the early assays as routine. Difficulties associated with early quantitative PCR techniques included: (i) ensuring that the PCR was within the linear range of amplification (that portion where the PCR signal is directly proportional to the input c...