Aging Aging

98 Engel, Adibzadeh, and PawelecPCR primers consist of 10-base arbitrary primer and the same locking primerused in the first strand synthesis (T 11 VN; V can be A, G, or C; N can be any ofthe four nucleotides). The process of the DDRT-PCR is the following:1. During the first strand synthesis, the locking primer samples a sizeable portion ofthe mRNA (potentially 1/16 th ) present in the cell.2. The arbitrary primer permits amplification of reverse-transcribed cDNA speciesusing the locking primer.3. Repeated PCR cycling with low-stringency results in amplification of productsof a size 50°C). By structuring the PCR in this manner, theamplification of genomic DNA (gDNA) should be minimized because thereshould be a limited chance of incorporating the primer used in the first strandsynthesis into both ends of complementary portions of gDNA. In contrast, the18-base arbitrary primer is incorporated into one end of the cDNA as a result ofthe reverse transcription of the RNA and into the other end during the singleround of low-stringency PCR. Consequently, the authors conclude that the signalshould be derived from RNA, not from gDNA. The process of the RAP-PCR is the following:1. During the first strand synthesis, the arbitrary primer anneals and extends fromsites contained within the mRNA.2. Second strand synthesis proceeds in a similar manner during the single round oflow-stringency PCR.3. PCR amplification at high stringency proceeds strongly, having incorporated thearbitrary primer into both ends of the cDNA.4. A template-dependent competition exists that determines which potential PCRproducts will ultimately predominate. PCR products can be analyzed byacrylamide gel electrophoresis.Any primer of 18 bases can be used in this technique and the PCR productscan be larger than 500 basepairs.Here we present a detailed protocol that uses a poly(T) primer in the reversetranscriptase reaction and only one 18-base primer in the arbitrary amplificationof cDNA. The advantage of using the T12AG primer in the reverse transcriptionis the amplification of mRNA throughout total RNA. Previousexperiments have shown that the RAP-PCR principle with one primer in the