10.1. ISOLATION OF RNA One of the most important factors in generating high-quality full-length cDNA is the purity and integrity of the total RNA used as starting material. Chaotropic agents such as guanidinium chloride and guani-dinium isothiocyanate are capable of dissolving cellular structures and proteins, causing nucleoproteins to dissociate rapidly from nucleic acids and inactivating RNAse enzymes. Several procedures using chaotropic and reducing agents for the isolation of RNA from cells and tissues have been reported in the literature. One procedure commonly used to isolate intact RNA from all types of tissues, even those rich in RNAse enzymes, was first reported by Chirgwin et al. (34). Improvements to this method that allow sufficient amounts of RNA to be isolated from a number of samples in several hours have been published. Using the method of Chomcznski and Sacchi (35), it is possible to isolate sufficient RNA for reverse transcription from as few as 1 x 103 cells. RNA isolation kits that employ these and other improvements are commercially available from many of the molecular biology companies. One of the most popular of these commercial reagents is TRIzol (from Invitrogen, http://www.invitrogen.com), which is based directly on the method of Chomcznski and Sacchi (35). RNA can be further purified by oligo(dT) selection. This step is not typically necessary in RT-PCR applications but might help simplify subsequent cDNA synthesis and amplification steps by removing genomic DNA contamination that can amplify along with the target cDNA during PCR. Genomic DNA can also be removed from RNA preparations prior to cDNA synthesis by treatment with DNAse I.
10.2. AMPLIFICATION OF RNA Reverse transcriptase-PCR is an excellent method for analysis of RNA transcripts, especially for measuring low-abundance species or working with limited amounts of starting material. Classic blotting and solution hybridization assays require much more RNA for analysis and lack the speed and ease of technique afforded by PCR-based applications. RT-PCR couples the tremendous DNA amplification powers of the PCR with the ability of RT to reverse-transcribe small quantities of total RNA (1 ng or less) into cDNA. Using total RNA rather that poly(A) purified RNA reduces the possibility of losing specific (rare) mRNAs during the purification process and allows the use of very small quantities of starting material (cells or tissues). Other advantages of RT-PCR include versatility, sensitivity, rapid turnaround time, and the ability to compare multiple samples simultaneously.
Reverse transcriptase-PCR is basically a four-step process: (1) RNA isolation, (2) reverse transcription, (3) PCR amplification, and (4) PCR product analysis. RNA is isolated from cells or tissue and used as a template in a reverse-transcription reaction that produces cDNA, which serves as a template for the PCR reaction. Reverse transcriptase (retroviral RNA-directed DNA polymerase) is the enzyme used to catalyze cDNA synthesis. The RT reaction consists of five components: (1) cDNA synthesis primer, (2) an appropriate RT buffer, (3) dNTPs, (4) RNA template (total RNA or mRNA), and (5) RT enzyme. There are several commercially available RT enzyme preparations that can be used in standard RT-PCR applications. These include RT from Moloney murine leukemia virus (MMLV) and avian myeloblastosis virus (AMV). More recently, recombinant derivatives of these RT enzymes have become available that offer advantages over the native enzymes. SuperScript III Reverse Transcriptase (from Invitrogen, http://www.invitrogen.com) is a mutant form of MMLV RT with increased thermal stability (half-life at 50°C of 220 min) and reduced RNAse H activity. Advanced enzyme preparations like these produce the highest yields and confer high specificity when gene-specific primers are employed. However, conventional RT-PCR applications employ oligo(dT) as the primer for RT. This type of primer is designed to bind specifically to the poly(A) tail of the mRNA, although these primers can also anneal to long stretches of adenosine within mRNA sequences. To combat this problem, anchored oligo(dT) primers have been generated by adding a single G, A, or C to the 3' end of the oligo(dT) (36). The PCR aspect of RT-PCR is identical to that described for DNA applications, with the exception that the template is cDNA rather than genomic DNA.
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