Viral RNA Extraction and Gag Gene Amplification

Viral RNA was extracted from plasma samples obtained from MSM subjects with the QIAamp Viral RNA Mini-kit (Qiagen, UK). The Gag gene was amplified using the SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China), followed by a second round of PCR with Gag-specific primers [36 (link)]. PCR products were gel purified with the QIAquick gel extraction kit (Qiagen, UK) and cloned with a TOPO TA cloning kit (Invitrogen, USA). The cloning PCR fragments were then sequenced with Sanger sequencing method using ABI 3730 DNA analyzer by Huada Genomics Company (China). A sequencer program was used to assemble and edit individual sequence fragments.

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Jiang F., Han X., Zhang H., Zhao B., An M., Xu J., Chu Z., Dong T, & Shang H. (2016). Multi-layered Gag-specific immunodominant responses contribute to improved viral control in the CRF01_AE subtype of HIV-1-infected MSM subjects. BMC Immunology, 17(1), 28.

Publication 2016

QIAamp Viral RNA Mini-kit SuperScript Polymerase One-Step RT-PCR System QIAquick gel extraction kit TOPO TA cloning kit

Gag gene Plasma Primers Rt pcr Topo Viral rna

Corresponding Organization :

Other organizations : National Health and Family Planning Commission, China Medical University, University of Oxford, John Radcliffe Hospital, MRC Human Immunology Unit, Medical Research Council

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Variable analysis

independent variables

QIAamp Viral RNA Mini-kit (Qiagen, UK) used for viral RNA extraction from plasma samples
SuperScript Polymerase One-Step RT-PCR System (Takara, Dalian, China) used for Gag gene amplification
Gag-specific primers used for the second round of PCR
TOPO TA cloning kit (Invitrogen, USA) used for cloning PCR fragments
Sanger sequencing method using ABI 3730 DNA analyzer by Huada Genomics Company (China) used for sequencing the cloning PCR fragments

dependent variables

Gag gene sequence data obtained from the cloning PCR fragments

control variables

Plasma samples obtained from MSM (men who have sex with men) subjects

positive controls

None specified

negative controls

None specified

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