Measuring Hepatitis C Viral Load Using Quantitative Reverse Transcription Polymerase Chain Reaction

Begin with total cellular RNA containing Hepatitis C virus or HCV RNA.
Take the known copies of HCV RNA as a standard.
Add a buffer containing reverse transcriptase, DNA building blocks, HCV-specific primers, and initiate reverse transcription to synthesize complementary DNA or cDNA. The viral RNA is degraded afterward.
Add a buffer containing HCV-specific primers, DNA polymerase, DNA building blocks, and a fluorescent dye.
Initiate quantitative PCR thermal cycling.
The primer and DNA polymerase bind to single-stranded DNA and synthesize double-stranded DNA or dsDNA.
The dye binds to the dsDNA, producing a detectable fluorescence signal.
Later, heating separates the DNA strands, cooling allows primer binding, and new double-stranded DNA is synthesized.
As cycles repeat, dsDNA copies increase exponentially, producing higher dye fluorescence.
Compared with the standard, an early fluorescence rise indicates high viral load, while a delayed rise indicates low viral load in the infected cells.

Reverse transcribe one microgram of total cellular RNA using reverse transcriptase enzyme and a specific primer for the HCV sense strand that binds to the five prime untranslated region in a 0.2 milliliter