Materials and Reagents

1. HepG2/C3A human hepatoma cell line (ATCC, catalog number: CRL-10741 )
   
2. Huh7 human hepatoma cell line (Japanese Collection of Research Bioresources, catalog number: JCRB0403 )
   
3. HEV Kernow-C1 p6 plasmid (genotype 3 full-length genome; a kind gift from Suzanne Emerson, NIH) (Shukla et al., 2012)
   
4. MluI restriction endonuclease with accompanying 10x buffer D (Promega Corporation, catalog number: R6381 )
   
5. QIAquick gel extraction kit (QIAGEN, catalog number: 28704 )
   
6. T7 RiboMAX large scale RNA production system (Promega Corporation, catalog number: P1300 )
   
7. RNeasy mini kit (QIAGEN, catalog number: 74104 )
   
8. ScriptCap m7G capping system (CELLSCRIPT^TM, catalog number: C-SCCE0610 )
   
9. Anti-fungal agent (2.5 µg/ml, Fungizone) (Life Technologies, catalog number: 15290-018 )
   
10. Dulbecco’s modified Eagle’s medium with high glucose (DMEM) (Life Technologies, catalog number: 41965-039 )
    
11. Fetal bovine serum (FBS) (not heat-inactivated) (Life Technologies, catalog number: 10270-106 )
    
12. Opti-MEM I reduced serum medium (Life Technologies, catalog number: 31985-062 )
    
13. Lipofectin transfection reagent (Life Technologies, catalog number: 18292-011 )
    
14. Dulbecco’s phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Life Technologies, catalog number: 14190-094 )
    
15. Penicillin/Streptomycin (PS) (10,000 IU/ml) (Life Technologies, catalog number: 15140-148 )
    
16. CellTiter 96 AQueous MTS reagent powder [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega corporation, catalog number: G1111 )
    
17. Phenazine methosulphate (PMS) (Sigma-Aldrich, catalog number: P9625 )
    
18. Minimum essential medium (MEM) (no glutamine, no phenol red) (Life Technologies, catalog number: 51200-046 )
    
19. NucleoSpin RNA virus kit (MACHEREY-NAGEL, catalog number: 740956 )
    
20. One step qRT-PCR MasterMix Low Rox for probe assays (Kaneka Corporation, Eurogentec, catalog number: RT-QPRT-032XLR )
    
21. Forward primer (5’-GGTGGTTTCTGGGGTGAC-3’) and reverse primer (5’- AGGGGTTGGTTGGATGAA-3’) (custom order) (Integrated DNA Technologies) (dissolved to a final concentration of 10 µM in buffer TE)
    
22. Fluorescent probe (5’-FAM-TGATTCTCAGCCCTTCGC-MGBNFQ-3’ with FAM, 6-carboxyfluorescein; MGBNFQ, minor groove binder non-fluorescent quencher) (Life Technologies, custom order) (dissolved to a final concentration of 10 µM in buffer TE)
    
23. qPCR DNA standard
    The cloned cDNA target sequence is ligated into a plasmid (e.g. with the CloneJet PCR cloning kit, Thermo Fisher Scientific, catalog number: K1231 ) and miniprepped.
    Note: The concentration is determined by spectrophotometry and a logarithmic dilution series is made in buffer TE over 6 orders of magnitude. Alternatively, the original Kernow-C1 p6 plasmid can be used to make such a dilution series.
    
24. Tris (hydroxymethyl) aminomethane powder (Trizma base) (Sigma-Aldrich, catalog number: T1503 ) (dissolved to 1 M and pH 8.0 in H₂O)
    
25. Ethylenediaminetetraacetic acid disodium salt solution (EDTA, 0.5 M) (Sigma-Aldrich, catalog number: E7889 )
    
26. MTS/PMS solution (see Recipes)
    
27. Buffer TE (see Recipes)
    

Equipment

1. Spectrophotometer to determine RNA concentrations (e.g. Thermo Fischer Scientific, NanoDrop, model: ND-1000 )
   
2. Falcon transparent 6-well plate (Corning Incorporated, catalog number: 353046 )
   
3. Falcon transparent 96-well plate (Corning Incorporated, catalog number: 353072 )
   
4. 37 °C and 35 °C 5% CO₂ cell culture incubators
   
5. Centrifuge with temperature control
   
6. Saphire² microplate reader for absorbance (Tecan Trading AG)
   
7. PCR plate 96-well (SARSTEDT AG, catalog number: 72.1981.202 )
   
8. MicroAmp optical adhesive film (Life Technologies, catalog number: 4311971 )
   
9. ABI 7500 fast real-time PCR system (Life Technologies, catalog number: 4351107 )
   

Procedure