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

Özet

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.

Protokol

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-HCV RNA of known genome copies (10¹ to 10⁹ standard) using HCV sense strand primer.
   3. Carry out qPCR by using 50 ng of the resulting transcribed cDNA (complementary deoxyribonucleic acid) using specific HCV primers (JFH (Japanese fulminant hepatitis) RTQ F: 5’CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5’CCTATCAGGCAGTACCACA-3’) and DNA-binding green dye containing qPCR super mix. Perform qPCR for housekeeping gene PPIG (Peptidylprolyl isomerase G) as well (primers PPIG F: 5’-GAAGAGTGCGATCAAGAACCCATGAC-3’; PPIG R: 5’-GTCTCTCCTCCTTCTCCTCCTATCTTT-3’)
      NOTE: For calculating accurate intracellular HCV RNA level across samples, use cellular housekeeping gene, PPIG, expression level (Ct cycle) for normalization. Use the copy number of the FNX-HCV genome as the standard for the copy number determination.
   4. Use the following conditions when running qPCR to determine HCV RNA copy number: 95 ºC for 15 sec and 60 ºC for 30 sec (40 cycles) using a real-time PCR system.

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
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