Reverse transcriptase isolation, polymerase chain reaction

Although a number of mammalian retrotransposons have been discovered by chance, a variety of approaches have been developed specifically to survey the genome for the presence of retrotransposons as well as to facilitate their isolation and characterization. Of the three methods outlined here in detail, two (conserved restriction sites and phylogenetic screening) could lead to isolation of all three types of retrotransposons, whereas the third method [polymerase chain reaction (PCR) amplification of reverse transcriptase] will exclude the SINEs. An unrelated method that has been particularly useful for isolation of SINEs7 will not be detailed. 

Isolation of Reverse Transcriptase-Encoding Elements Using Polymerase Chain Reaction... 

Enzyme-linked immunoabsorbent assays (ELISAs) for VHP infections can be performed on samples inactivated by treatment with P-propiolactone. Reverse transcriptase polymerase chain reaction (RT-PCR) tests on samples following RNA extraction using chloroform and methanol can detect most of the VHP agents rapidly. RT-PCR is particularly useful when isolation of the virus is difficult or impractical, and was effective in detecting the agent causing HPS months before it was isolated in culture (48). 

An introduction to single gene transcript quantification methods. A number of methods are now available for the detection of gene activity via messenger RNA (mRNA). In all protocols, the first step is to isolate total RNA. This can be probed directly or used for reverse transcriptase polymerase chain reaction (RT-PCR)... 

Cells were washed with ice-cold PBS and total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (27). 5 pg total RNA was reverse-transcribed (RT) into cDNA using Moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo (dT)i primer by incubating the reaction mixture (15 pL) at 37 C for 90 min. The polymerase chain reaction (PCR) was performed as described previously (22). A final volume of 25 pL contained dNTPs (each at 200 pM), IX reaction buffer, 0.4 pM each primer (iNOS forward ... 

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