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

Abstract

Source: Ren, S., et al. A Protocol for Analyzing Hepatitis C Virus Replication. J. Vis. Exp. (2014).

This video demonstrates the procedure for quantifying Hepatitis C viral RNA using quantitative reverse transcription PCR to measure viral load in infected cell cultures.

Protocollo

1. Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR) for Assessing the HCV (Hepatitis C virus) Genome Copies 
   1. Perform a two-step RT-qPCR to determine the HCV genomic RNA (ribonucleic acid) copy number.
   2. Reverse transcribe 1 μg of total cellular RNA using reverse transcriptase and a primer specific for HCV sense strand that binds to 5’NTR (5′ nontranslated region) (JFH RTQ R: 5’CCTATCAGGCAGTACCACA-3’) or a primer specific for housekeeping gene peptidylprolyl isomerase G (PPIG R: 5’-GTCTCTCCTCCTTCTCCTCCTATCTTT-3’). Also, reverse transcribe FNX-H....

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
SUPERSCRIPT III RT	Life Technologies	18080085
SYBR QPCR SUPERMIX W/ROX	Life Technologies	11744500
ViiA 7 real-time PCR system	Life Technologies	NA
Renilla Luciferase Assay System kit	Promega	E2810
RNase-Free DNase	Promega	M6101
Primers