QRT-PCR

Keywords Barbus graellsii, Biomarkers, Choline esterases, Cyprinus carpio, Daphnia magna, Dreissena polymorpha, EROD, Metallothionein, qRT-PCR, Xenobiotics... 

List of Abbreviations cDNA, complementary DNA ddH20, double-distilled H2O dNTP, deoxyribonu-cleotide triphosphate EDTA, ethylenediaminetetraacetic acid MgCl2, magnesium chloride mRNA, messenger ribonucleic acid NaOH, sodium hydroxide PCR, polymerase chain reaction qRT PCR, quantitative reverse transcriptase polymerase chain reaction RNase, ribonuclease RT PCR, reverse transcriptase polymerase chain reaction UTR, untranslated region... 

Quantitative RT PCR (qRT PCR) can be used to accurately determine the levels of messages within given preparations of RNA. qRT PCR thermocyclers provide rapid online detection and quantification of mRNA, however, the initial purchase cost and the cost of reagents may be prohibitive for some laboratories. Methods of semiquantitative RT PCR have been used and good descriptions of these techniques are available (Samhrook and Russell, 2001). However, the same cDNA populations should not be used for differential display reactions and verification that a potential differential display band represents a differentially expressed gene. For this reason, independent cDNA samples should be prepared if both the screening and verification methods rely on PCR. qRT PCR, therefore, should be used in conjunction with other methods to verify that a differential display band represents a differentially expressed gene. 

We also investigated the prevalence of the gene transcript for collagen 1 as measured by qRT-PCR on tissue harvested on days 7, 21, and 48-55 after implantation. These data revealed a marked increase in type 1 collagen mRNA, specifically an approximately 10-, 30-, and 5-fold increase at these time points. These results suggest... 

Assimilable organic carbon (AOC) was determined in water using flow-cytometric enumeration and a natural microbial consortium as inoculum.134 Two bacterial species were used for the measurement of AOC in water, based on their respective 16S rDNA sequences. The AOC content in 41 water samples was determined with these two sets by quantitative real-time polymerase chain reaction (qRT-PCR).135... 

Another important set of multiplexed assays monitor mRNA transcript levels. The expression level of all the genes involved in a known signal transduction pathway or other selective genes can be monitored simultaneously as a way of following compound effects on a cell. The current technologies for multiple mRNA detection include quantitative reverse transcriptional PCR (qRT-PCR), qNPA (quantitative nuclease protection assays), mass array assay technologies and branched DNA detection on Luminex beads (Panomics). The applications of such multiplexed in vitro and cell-based detection systems should provide more predicative information in hit finding and lead characterisation. 

Figure 2. Coronin 1C in melanoma. A) Immunohistochemical staining of the indicated tissue samples for Coronin 1C. S m sections from melanoma tumors were stained with a new Coronin 1C specific mAb (Roadcap and Bear, unpublished reagent) for 30min at 37°C with a steam heat-induced epitope retrieval, using the streptavidin/biotin method with an alkaline phosphatase label and Permanent Red (Dako) as the chromogen and then stained with hematoxylin. B) qRT-PCR analysis of the indicated melanoma cell lines treated with the Erk inhibitor U0126 or vehicle control for 24 hours.Dotted line indicates lx threshold.

Fig. 6 Methionine synthase mRNA levels in human cortex are reduced in autism. RNA samples from autistic and non-autistic subjects were probed using qRT-PCR with specific primers to the CAP and COB domains of methionine synthase, n = 11 for each group ( p<0.05 compared to control for the CAP primer set p<0.05 compared to control for the COB primer set)...

Amary, M., Berisha, F., Bernard , F. et al. (2007) Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms analysis of conventional RT-PCR, qRT-PCR... 

Time course analysis of gene expression levels using QRT-PCR in the acute phase of the penetrating ballistic brain injury between 3 and 6 h indicates an upregulation in the expression of cytokines TNF-a (eightfold to 11-fold), IL-ip (11-fold to 13-fold), and IL-6 (40-fold to 74-fold) as well as the cellular adhesion molecules VCAM (twofold to threefold), ICAM-1 (sevenfold to 15-fold), and E-selectin (11 -fold to 13-fold). These processes are consistent with the upregulation... 

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