Quantitative PCR

QA/QC Control Metrics QT Prolongation Quantification of Diug Effect Quantitative PCR Quinolinic Acid Quinolones... 

Tanabe, S., Hase, M., Yano, T., Sato, M., Pujimura, T., and Akiyama, H. (2007). Real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Biosci. Biotechnol. Biochem. 71, 3131-3135. 

Detection of miRs by reverse transcription (RT) and quantitative PCR (qPCR)... 

K Withby, J Garson, A Baret. A microtiter format quantitative PCR assay for HCV RNA employing xanthine oxidase generated chemiluminescence. Proceedings of... 

Arya, M., Shergill, I.S., Williamson, M., Gommersall, L., Arya, N., and Patel, H.R. 2005. Basic principles of real time quantitative PCR. Expert Review of Molecular Diagnostics 5, 209-219. 

Quantitative PCR has been widely used to determine the amount (number of molecules) of DNA molecules in a test sample. The best quantitative PCR method involves the addition of known amounts of a similar DNA or RNA fragment, such as one containing a short deletion or specific mutation, to the test sample before amplification. Such internal standards must be precisely calibrated to ensure that they are amplified and detected in a form and manner that are similar to the test sample. The ratio of the internal standard and the targeted template will depend on the amount of internal standard added and allows for the determination of the amount of the targeted molecule in the test sample. Therefore, the ideal standard for quantitative amplification based assays should have a structure that is comparable to the template of interest and which allows for the simultaneous amplification of both template and standard using a single primer pair. 

Buck KJ, Harris RA, Sikela JM. 1991. A general method for quantitative PCR analysis of mRNA levels for members of gene families application to GABAa receptor subunits. Biotechniques 11 636-641. 

Ginzinger DR. 2002. Gene quantification using real-time quantitative PCR an emerging technology hits mainstream. Exp Hematol 30 503-512. 

To obtain accurate results in qMSP/QM-MSP analyses, the best conditions have to be worked out to achieve reliable standard curves during the test trials. This could be achieved with the use of unmethylated and methylated templates such as placental DNA and in vitro methylated DNA, respectively, and by performing bisulfite treatment as described. To find the best condition for each gene, purify the DNA, calculate the concentration and copy numbers, and dilute the DNA sequentially (e.g., 1,1/2,1/4,1/8, 1/16, 1/32, and 1/64) and perform real-time PCR with several dilutions of the primers (e.g., 25, 50, 75ng each in various combinations). For example, you should see the amplification plots as indicated in Fig. 9.4 with 50% (Fig. 9.4A) or 25% (Fig. 9.4B) sequential dilution. These test trials and any other quantitative PCR (qPCR)ZQM-MSP experiments need to be done in duplicate or triplicate to ensure that the required skills and instruments for equal pipeting are in place, or the impacts could be minimized by averaging the results of the triplicate experiments. Similar to MSP, for each qMSP or QM-MSP trials use placental and... 

To further confirm the data, we performed Y chromosome PCR. The sensitivity of this method was 10 or 1000 cells detected in a total of 10 cells that were screened. Organs of female rats infused with male MSC were harvested at 24 hand at day 3 after infusion. Real-time quantitative PCR withY chromosome-specific primers showed amplification only in the lungs at 24 h after cell infusion (Figure 5), whereas kidney, liver, spleen, and bone marrow were negative, thereby further corroborating our results about a clearance of MSC from kidneys long-term. 

Luchi N etal., A real-time quantitative PCR assay for the detection of Sphaeropsis sapinea from inoculated Pinus nigra shoots, JPhytopathol 153 37—42, 2005. 

Assess the downregulation of your target in the presence or absence of doxycycline by Western blot or quantitative PCR (qPCR) (see Note 21). 

In 1996, after eight years in the planning stage, a quantitative PCR assay for HIV-1 in human plasma was released by Roche Molecular Systems. This test, the result of intensive efforts by John Sninsky and his team at Roche, allowed quantitation of HIV-1 viral load to 400 viral copies (just 200 viral particles, as each HIV-1 virion contains two copies of the virus). 

An international effort is underway to harmonize methodologies for detecting BCR-ABLl transcripts and kinase domain mutations and adjusting the results so they are standardized from one lab to another (28,29). The availability of a reproducible real-time quantitative PCR (RQ-PCR) has shown in one study that patients with a major molecular response do not have evidence of cytogenetic abnormalities in their bone marrow, and therefore a policy of performing bone marrow biopsies only in patients who have not achieved or have lost a major molecular response would allow many patients to forego the discomfort and expense of multiple bone marrow biopsies (30). 

Branford S, Rudzki Z, Parkinson I et al. Real-time quantitative PCR analysis can be used as a primary screen to identify patients with CML treated with imatinib who have BCR-ABL kinase domain mutations. Blood 2004 104 2926-2932. 

It has been demonstrated that the human myeloid zinc finger (MZF1) (92) functions as a transcription repressor regulating ERCCl transcription in response to cisplatin-induced DNA damage. It was found that MZFl mRNA is constitutively expressed in human ovarian cancer cells. Quantitative PCR in the same cells showed that MZFl mRNA was decreased upon cisplatin exposure (93). Therefore, decreased expression of MZFl is a mechanism to be further investigated. 

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