![]() 4 – 8 This may not only affect the run of the samples in real-time RT-PCR but also may affect the generation of the standard curve. First, despite the luxury of being able to choose the method of priming (gene specific, random hexamer, or oligo dT), different methods of priming in the RT step have been shown to provide different sensitivities and efficiencies. ![]() However, there are other factors involved with each method that might produce differing real-time results. Typically, scientists choose one-step or two-step methods based on these factors. This allows for the ability to convert all the messages in an RNA sample into cDNA, which would allow for archiving of samples and future testing of other genes. The main advantage to two-step RT-PCR is that typically random hexamer or oligo dT primers are used in an RT reaction in a separate tube. The RNA from the original sample must be initially aliquoted for archival storage and future testing. With the one-step method, gene-specific primers are used and both the RT and PCR occur in one reaction tube therefore, other genes of interest can not be amplified for later analysis. The advantages to one-step real-time RT-PCR is that it is quicker to set up, less expensive to use, and involves less handling of samples, thereby reducing pipetting errors, contamination, and other sources of error. There are advantages and disadvantages to both systems. ![]() The other method involves creating cDNA first by means of a separate reverse transcription reaction and then adding the cDNA to the PCR reaction (two-step). One method involves including the RT step into the same tube as the PCR reaction (one-step). 1 – 3 However, there are two primary ways that real-time RT-PCR can be carried out. Real-time, or quantitative reverse transcription polymerase chain reaction (RT-PCR), has become an increasingly popular technique for the analysis of gene expression. However, using the one-step method with gene-specific priming may be more sensitive for quantification of certain genes such as PolR2A. ![]() In summary, both SuperScript III one-step and two-step methods yield reaction efficiencies close to 100% and produce similar, accurate, linear standard curves. However, for the lesser expressed PolR2A mRNA there was a 5 cycle lower threshold for one-step. The sensitivities of one-step and two-step methods, as measured by cycle threshold values, were similar for GAPDH and B2M. Reaction efficiencies ranged from 97.7 ± 0.9% to 99.4 ± 1.8% for one-step and 98.0 ± 0.2% to 102.6 ± 1.3% for two-step RT-PCR (R 2 values for all reactions ≥ 0.995). Reaction efficiencies were determined by generating standard curves using total RNA isolated from human skeletal muscle and brain. In this study, real-time reactions were set up using the housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), β 2-microglobulin (B2M), and RNA polymerase 2 subunit A (PolR2A). There has been little research conducted to test if SuperScript III quantitative one-step (reverse transcription carried out in the same tube as PCR) and two-step (reverse transcription carried out in a separate reaction) RT-PCR systems provide similar real-time results. Real-time reverse transcription polymerase chain reaction (RT-PCR) is a commonly used technique to analyze gene expression. ![]()
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