Reverse-transcribed polymerase chain

Niesser M, Volk BA. Altered Thl/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR). Clin Exp Immunol 1995 101 428 35. 

A popular technique used to observe gene expression is reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR works by reverse transcribing mRNA to a complementary DNA sequence that corresponds to the gene of interest. Several years ago, Zabzdyr and Lillard showed that CE could be used to separate and detect RT-PCR products corresponding to P-actin in individual... 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

Figure 2 Cytokine gene expression in immunopotentiating reconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). PBMC were cultured in the presence or absence of IRIV. On days 1 and 2, culture cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNAs thus obtained were tested in real time polymerase chain reaction assays in the presence of primers specific for the indicated cytokine genes. Source From Refs. 6 and 9.

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 ... 

To be converted into an active compound, all the NRTIs have to be phosphorylated intracellularly to the triphosphate form via monophosphates and diphosphates. The triphosphates compete with cellular nucleotides and inhibit HIV reverse transcriptase by introducing a chain terminator into the growing complementary DNA strand during reverse transcription. However, this mechanism also occurs when human DNA is transcribed by the human DNA poljmerase. In fact, the first NRH in clinical use (zidovudine) was initially developed as an anticancer drug, targeting human DNA polymerase in cancer cells. However, aU NRTIs have higher specificity for viral reverse transcriptase than for the human DNA polymerase. 

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