One-Step RT-PCR

To analyze RNA by PCR an additional step has to be introduced to transcribe RNA into cDNA, which is a more stable single-strand DNA complementary to the targeted RNA. This can be achieved by the reverse transcription (RT) of the template RNA using a reverse transcriptase enzyme. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers for the reverse transcription reaction, whereas in two-step RT-PCR random primers can be used. 

In one-step RT-PCR, both cDNA synthesis on the RNA temple and the cDNA 

Reverse transcriptase, DNA polymerase enzyme with buffer. The following enzymes are the most common used enzymes for one-step RT-PCR  

Primers site-specific primers selective for the mRNA template and PCR primers. 

Primer extension at 68 °C for 1 min all three phases 2-4 are repeated for ten cycles. 

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Superscript III one-step RT-PCR system with Platinum Taq polymerase (Invit-rogen) and MJ Research PTC200 Peltier Thermal cycler for amplification. 

Stellrecht KA, Harding I, Hussain FM, Mishrik NG, Czap RT, Lepow ML, et al. A one-step RT-PCR assay... 

Nikiforova MN, Groen P, Mutema G, et al. Detection of SYT-SSX rearrangements in synovial sarcomas by real-time one-step RT-PCR. Pediatr Dev Pathol. 2005 8 162-167. 

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. 

Titan one tube single-step RT-PCR system (Boehringer Mannheim, Cat. No. 1888 382)... 

By first making a DNA copy of an RNA molecule, one can also amplify RNA sequences. Because reverse transcriptase (copies RNA to DNA) is used in the first step, this is called RT-PCR. 

Proper fixation is one of the most critical steps in an in situ RT-PCR or in situ hybridization experiment, because each tissue type must have optimized fixation conditions. Particularly archival tissues may require individual specific treatment in order to meet with in situ experimental requirements. Errors in fixation will only be discovered after the entire hybridization process has been completed (see Note 10). 

DEPC-treated water should be used for all RNA preparation solutions, gloves should be worn at all times, and RNase-free tips and tubes used. It is not necessary to purify mRNA, since total RNA prepared from freshly isolated or even frozen (liquid nitrogen or-80°C) lymphocytes is pure enough to carry out the RT-PCR reactions. A number of commercial kits are now available for RNA isolation (e.g., Stratagene), and use of one of these is recommended. Alternatively the single-step guanidimum thiocyanate/phenol extraction method of Chomczynski and Sacchi (23), on which most kit protocols are based, should be employed. If the lymphocyte numbers are low (<5 x 106), carrier RNA (100 pg of 16S ribosomal RNA) can be added at the start of this procedure to aid recovery. The preparation ends with a precipitation step using isopropanol, and the RNA may stored at -20°C in this form until required. 

One of the first steps to go behind the metastatic cascade is the examination of peripheral blood to detect potential circulating malignant cells. This method is useful to follow-up malignancies spreading via the blood stream such as sarcomas, melanoma, neuroblastoma, prostatic, thyroid and hepatocellular carcinomas. PCR-and RT-PCR-based molecular techniques are found to be the most efficient assay to detect circulating tumor cells in peripheral blood, and allow the detection of one tumor cell in up tolO background nucleated blood cells. However, it is important to consider that the presence of tumor cells in peripheral blood is only one step in a multistep process and not all tumor cells circulating in peripheral blood are able to... 

A further important step in quantitative RT-PCR is the production of a single-stranded (ss) complementary DNA copy (cDNA) of the RNA through the reverse transcriptase (RT). Its dynamic range, sensitivity, and specificity are of prime consideration for a successful kinetic RT-PCR assay. For many quantitative applications, moloney murine leukaemia virus (MMLV) RT is the enzyme of choice, as its cDNA synthesis rate is up to 50-fold greater than that of avian myeloblastosis virus (AMV). Newly available thermostable enzymes maintain their activity up to 70°C, thus permitting increased specificity and efficiency of first primer annealing. Fiowever, this enzyme type may be less robust than more conventional ones as it appears to be more sensitive to inhibitors present in RNA preparation. 

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