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 tube. Also, reverse transcribe F-N-X-H-C-V-R-N-A of known genome copies using an HCV sense strand primer using a real-time PCR system.

Carry out QPCR by using 50 nanograms of the resulting transcribed cDNA using specific HCV primers and DNA binding green dye containing QPCR super mix. Use the following conditions when running QPCR to determine the H-C-V-R-N-A copy number