Reverse transcription polymerase chain reaction (RT-PCR) is a core molecular technique designed to detect and quantitate total RNA or messenger RNA (mRNA), with a high degree of sensitivity and accuracy. In this modified version of the standard PCR process, mRNA, which serves as the initial template, is first reversed transcribed to complementary DNA (cDNA) and then subsequently amplified via PCR for downstream analysis. Relative to other techniques for measuring mRNA, such as Northern blot analysis, RNAse protection assays, or in situ hybridization, RT-PCR is significantly more robust at detecting the RNA transcript of any gene regardless of its relative abundance. Consequently, RT-PCR is widely used to quantitatively study gene expression, examine transcript variants, and generate cDNA templates for cloning and sequencing. Furthermore, RT-PCR has become an instrumental diagnostic tool for the detection of pathogens, including viruses that cause Ebola, HIV and the novel coronavirus disease (Covid-19).
Table 6. Recommended FRET pairs for developing FRET oligonucleotides
Donor \ Acceptor | DABCYL | TQ1 | TQ2 | TQ3 | TQ4 | TQ5 | TQ6 | TQ7 |
EDANS | +++ | +++ | + | – | – | – | – | – |
MCA | +++ | +++ | + | – | – | – | – | – |
Tide Fluor™ 1 | +++ | +++ | + | – | – | – | – | – |
FAM FITC |
+ | + | +++ | + | – | – | – | – |
Cy2® Tide Fluor™ 2 |
+ | + | +++ | + | – | – | – | – |
HEX JOE TET |
– | – | + | +++ | + | – | – | – |
Cy3® TAMRA Tide Fluor™ 3 |
– | – | + | +++ | + | – | – | – |
ROX Texas Red® |
– | – | – | + | +++ | + | – | – |
Tide Fluor™ 4 | – | – | – | + | +++ | + | – | – |
Cy5® Tide Fluor™ 5 |
– | – | – | – | + | +++ | + | – |
Cy5.5® Tide Fluor™ 6 |
– | – | – | – | – | + | +++ | + |
Cy7® Tide Fluor™ 7 |
– | – | – | – | – | – | + | +++ |