反轉(zhuǎn)錄PCR原理步驟小結(jié)

1、RT-PCR for Human Tissue1Principle of Reverse Transcription-Polymerase Chain Reaction (RT-PCR)RT-PCR is used to clone expressed genes by reverse transcribing the RNA of interest into its complementary DNA (cDNA) through the use of reverse transcriptase. Subsequently, the newly synthesized cDNA is amp

2、lified using traditional PCR.General ProtocolGeneral Principle2RNA Extract & QuantificationHumanTissueTissue RNA PrepmateTM used for RNA extrationQubit Fluorometer & Quant-iT RNA Kit used for quantification3Protocol of Reverse Transcription-Polymerase Chain Reaction (RT-PCR)In the one-step protocol,

3、 the components of RT and PCR are mixed in a single tube at the same time. The one-step protocol generally works well for amplifying targets that are reasonably abundant.Alternatively, RT-PCR can be done in two steps, first with the reverse transcription and then the PCR. The two-step protocol is us

4、ually more sensitive than the one-step method; yields of rare targets may be improved by using the two-step procedure.We recommend the two-step protocol for this class.4Most mRNA contains a long stretch of adenosine residues (AAAn) at the 3 end of the message; therefore, investigators anneal a prime

5、r consisting of 15 deoxythymidine residues (dT15) to the 3 end of the message. Reverse transcriptase then transcribes a complementary DNA strand, starting at the dT15 primer. The cDNA can be isolated by raising the pH of the solution, thereby denaturing the double-stranded hybrid and cleaving the RN

6、A. Principle of cDNA PreparationReverse TranscriptionProcedures of cDNA PreparationAssemble the reaction appropriate in a 0.25 ml PCR tube including:RNA templateNonspecific primerReverse transcriptaseDNA polymeraseBuffer reagentMix gently, spin briefly.Incubate in the thermacycler at:44C for 1 h92C

7、for 10 min to inactivate the reverse transcriptase.Store reaction at -20 or proceed to PCRNote to wear gloves and mask at all times to avoid RNase contamination.5Polymerase Chain Reaction (PCR)Principle of PCRCopyright 2012 by HIBELGAUFTS. All rights reserved.The first step of the PCR reaction separ

8、ates the double strands into single strands (strands 1 and 2) by heat denaturation. The second step is a process called primer annealing, i.e., the formation of partially double-stranded regions by interaction with the primer binding sites on each strand with the corresponding primer.DNA polymerase

9、is then used to extend the primer region and to synthesize full-length complementary strands (light and dark blue lines) of strands 1 and 2, yielding two new double strands 1-3 and 2-4. This reaction requires the presence of deoxynucleoside triphosphates, which serve as substrates for the polymerase

10、.The process of denaturation, primer annealing, and primer extension can be repeated with the new double strands 1-3 and 2-4 since they also contain the primer binding sites. The reaction products of this second reaction cycle are four double strands: 1-5, 2-7, 3-6, 4-8.By repeating the process of d

11、enaturation, primer annealing and primer extension some 20 to 30 times one obtains an exponential amplification of the DNA sequence located between the two primer binding sites. 6Primer PreparationSetting up negative controls:The minus-RT control from the previous step, or alternatively, untreated R

12、NA can simply be subjected to PCR.A minus-template PCR, it should have all the PCR components, but use water as template instead of an aliquot of the cDNA (RT reaction).Setting up positive controls:Perform PCR to amplify a cDNA that corresponds to a basal or housekeeping transcript.Use genomic DNA i

13、solated from S2 cells as template.Assemble the reaction appropriately including:RT reactionForward primerReverse primerdNTP mixTaq polymeraseMgCl2 solutiondH2OPCR buffer reagentIncubate in Thermacycler:Initial denaturation: 94C for 4 min30 cycles: Denature at 94C for 30 sec Anneal at 55C for 2030 sec* Extend at 72C for 45 sec *Final extension: 72C for 5 minProcedures of PCR*Start with the annealing temperature suggested by your primer design software.*The rule of thumb is to use a