qPCR技术.docxVIP

Template preparation: DNA or RNATrizol : column-based kit, guidelines differ due to different buffersClean and free from residual buffersMain problem: exaction of inhibitors together with the nucleic acids or degradation of sampleInhibitor found in blood or environmental samplesGeneral rule: fresh sample, -80 centigrade storedOne-step or two-step reaction?Total RNA, perform RT before qPCROne step/two steps: avoid contamination, RNase free, tubes containing the reaction be maintained on ice30min and 50 centigrade, if random primers/oligo d(T) used(two steps) initial 10 min-step at 25 centigrade to maximize the annealing efficiency as the short oligo d(T) have a lower TmStored -80 centigrade, limiting amount of starting material separate RT stepBack ground???Which type of primers for the Reverse Transcription?Short random primersOligo d(T) bind to the pply-A tail of the RNA ant then transcribe RNASpecific primersOne-step qrt-PCR: specific primers, avoid aspecific products RT at 40-50 centigrade specific PCR primers desingned at 60 centigradePartial annealing???Two-steps qrt-PCR: oligod(T) or/and random primers in RT stepSpecific primers in PCR stepQualitative vs. Quantitative PCRTaq polymerase , extension of short single-stranded primersRepeated cycles of heat de-naturation, primer annealing and primer extensionPrimers, specifically bind to extremities of the DNA fragment to be amplifiedTaqpolymerase use target DNA as a template for primer extensionEach cycle, more template DNA, exponential manner, doubling, until one of reagents limitingQualitative PCRDetection chemistry allows quantificationAmount at a certain point of the run is related to the initial amount of target in the sampleMost common applications: gene expression analysis, pathogen detection/quantification and microRNA quantificationInitially a exponential process, eventually a plateau phaseAssume the amount double each cycle and no bias due to limiting reagentsAnalysis: Ct value, the more