Real-time qPCR

Measuring Gene Expression Real-Time qPCR... 

This chapter summarizes our experience with this approach and describes some of the critical parameters involved in the preparation of rat neural tissue for LCM, with particular emphasis on protocols applied to the Arcturus Autopix LCM. This instrument has been superceded by more recent models, which are now marketed and distributed by MDS Analytical Technologies (http // www.moleculardevices.com). The principles described here, however, are applicable to tissue preparation for most LCM instruments. We describe two basic protocols for the isolation of samples of rat brain, first from unstained microdissected regions, and second, from brain cells that express specific antigens identified by immunostaining. In addition, we compare the effects of different fixation conditions on tissue recovery and RNA content using real-time QPCR. 

Refinements of PCR involve redncing the time it takes to prodnce snfficient DNA for reqnired pnrposes, redncing error rates, and reducing the amonnt of sample needed at either the beginning or the end of the PCR process. qPCR stands for quantitative PCR, and RT-qPCR stands for real time qPCR. The primers must be extremely specific to the template being amplified, and there is a limit to the maximum length of DNA that can be amplified. Any slight contamination with nonintended DNA can resnlt in an incorrect prodnct. 

Quantitative polymerase chain reaction, also called real-time RT-PCR or QPCR, is a method which employs insertion of a signal, such as fluorescence or enzyme activity, into PCR products generated by RT-PCR to determine the amount of messenger RNA (mRNA) in a tissue accurately. 

Badosa E, Trias R, Pares D, Pla M and Montesinos E. 2008. Microbiological quality of fresh fruit and vegetable products in Catalonia (Spain) using normalised plate-counting methods and real time polymerase chain reaction (QPCR). J Sci Food Agric 88(4) 605-611. 

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... 

Recently, real-time PCR (quantitative PCR, qPCR) has been used for identification of S. aureus species (Du et al. 2(X)2 Palomares et al. 2003) and the species of coagulase-negative staphylococci (Edwards et al. 2001). 

Quantitative PCR (qPCR) is a variation of real-time PCR that can be used to evaluate gene expression levels or gene copy numbers. Quantitative assessment of the initial template used for PCR amplification can be attained by comparing the amount of PCR product of the target sequence with the PCR products generated by amplification of the known quantities of DNA or cDNA. 

Finally, on-chip DNA quantitation will undoubtedly be an important feature of microfluidic sample processing for a number of different applications where the amount of input DNA for downstream genetic analysis is crucial. As discussed more extensively in Chapter 37, quantitative PCR (qPCR) methods in microfluidic systems have not been demonstrated to date. However, there are a number of publications describing real-time PCR in microdevices and it is likely only a matter of time before... 

Real-time PCR or quantitative polymerase chain reaction (qPCR), is a highly sensitive technique that can be used to quantify genes of interest (functional... 

Symbols NaOH, sodium hydroxide qPCR, real-time PCR SDS, sodium dodecyl sulphate (Juvonen, 2009, p. 273). 

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