Two-step transcriptional amplification

Zhang L, Adams JY, BiUick E, Hagan R, Iyer M, Le K, Smallwood A, Gamhhir SS, Carey M, Wu L. Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer. Mol Ther 2002 5 223 - 232. 

Iyer M, Salazar FB, Lewis X, Zhang L, Carey M, Wu L, Gambhir SS. Noninvasive imaging of enhanced prostate-specific gene expression using a two-step transcriptional amplification-based lentivirus vector. Mol Ther 2004 10 545-552. 

Iyer M, Wu L, Carey M, Wang Y, Smallwood A, Gambhir SS. Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci USA 2001 98 14595-14600. 

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. RT-PCR performed on fresh-frozen tissue provides high-quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplification. To attain a more stable RT-PCR amplification from FFPE tissues, it is typical to choose a target that is less than 150 to 200 nt long. 

Reverse transcriptase (RT) PCR (RT-PCR) can be used to detect RNA in specimens, especially ssRNA viruses. Using the ssRNA as a template, cDNA (complementary DNA) can be synthesized with the enzyme reverse transcriptase, which can further be used as a template in PCR amplification. In two-step RT-PCR, extracted RNAs are first mixed with RT and suitable primer to synthesize cDNA and then followed by adding Taq polymerase and PCR primer pairs for PCR amplification. In one-step RT-PCR, all the ingredients are mixed together and allowed for one cycle of reverse transcription to synthesize cDNA followed by 30 + cycles of PCR amplification. The product can be analyzed by agarose gel electrophoresis. Commercial RT-PCR test kits are available. 

With the addition of an initial reverse-transcriptase step to form cDNA from RNA in the sample, RNA targets can also be successlEuIly amplified into DNA copies. The reverse transcription and DNA amplification steps are usually catalyzed by two different polymerases, but some thermostable enzymes (such as the Tth polymerase) have both DNA polymerase and reverse transcription activities, so that both steps can be performed in the same tube with the same enzyme. 

When comparing hybridization results between two cell types, the same type and quantity of RNA should be used to generate both probes. If sufficient material is available, probes can be directly generated by incorporating fluorescent nucleotides during a reverse-transcription reaction. Otherwise an additional RNA amplification step will be needed. This is not desirable because it could cause problems with transcript representation. 

Extraction of DNA from biological samples can be accomplished by precipitation or affinity methods [15]. Amplification of the amount of DNA is also needed before any sequence detection can be done. This can be done by a method known as polymerase chain reaction or PCR in short. This process is depicted in Figure 8.6. Briefly, original double stranded DNA molecules, shown as black rectangles, are heated to more than 90°C for separation. Afterward, DNA primers, shown as squares, as well as nucleic acids are added to the solution to initiate the DNA synthesis, forming two pairs of double stranded DNA at the end of the first cycle. The process is repeated for more than 30 times, doubling the amount of DNA at each step [16]. RNA can also be amplified using a similar process known as reverse transcription-polymerase chain reaction (RT-PCR) [17]. 

No conclusive information is as yet available on the mechanism of the initial step of rDNA amplification. It is not known whether the first amplified copy of rDNA is a replica or an excision product. A process whereby amplification is mediated by RNA followed by reverse transcription has also been suggested (Tocchini-Valentini and Crippa, 1971 see later). Furthermore, it remains unknown whether one, a few, or all chromosomal rRNA genes are involved in gene amplification. Two different mechanisms—not necessarily mutually exclusive— have been proposed for the production of additional rDNA replicas (Miller, 1966). The amplified rDNA might be synthesized only on the chromosomal template. Alternatively, the newly synthesized rDNA copies could serve as new templates for subsequent replications, resulting in a cascade effect. Autoradiographic evidence has been presented which favors the second mechanism (Macgregor, 1968). 

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