HBV DNA was isolated from cells or supernatants by use of the QIAamp mini kit as previously described (39 ). Total cellular RNA was harvested from cells using the QIAshredder and RNeasy mini kits (QIAGEN, Valencia, CA, USA) as reported previously (35 (link), 39 , 42 (link)). RNA concentrations were determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Inc., DE, USA). cDNA was synthesized by reverse transcription using the High-Capacity cDNA Kit (Thermo Fisher Scientific). qPCR was performed using the Power Up SYBR Green Master Mix (Thermo Fisher Scientific) using the Quant Studio 3 platform qPCR machine (Thermo Fisher Scientific, Waltham, MA). The target mRNA expression was normalized to GAPDH using the 2-ΔΔCt method to obtain mRNA arbitrary units (fold-change). Primer sequences are listed in Table 1 .
Protocol detail
HBV DNA and RNA Isolation, Quantification, and Analysis
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Corresponding Organization : Harvard University
Other organizations : Hainan Medical University, University of Science and Technology of China, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing Medical University, Jiangsu Province Hospital, Southwest University, Chongqing Medical University, China Medical University, Soochow University, MedStar Georgetown University Hospital, Georgetown University, St Vincent's Hospital
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Variable analysis
independent variables
- RNA isolation method (QIAamp mini kit, QIAshredder, RNeasy mini kits)
- CDNA synthesis method (High-Capacity cDNA Kit)
- HBV DNA levels
- Total cellular RNA levels
- Target mRNA expression levels
- GAPDH mRNA levels (used for normalization)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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