Abstract
Experiment method
Sample collection, PBMC RNA extraction and RNA-seq sequencing
All participants were screened for HIV antigens and antibodies via standard ELISA analyses,and these findings were further confirmed by Western Blotting. CD4+ T-cell counts wereobtained by flow cytometry, and the viral loads were measured by qRT-PCR. HIV serumpositivepatients were also diagnosed for M. tuberculosis, as assessed by microbiology tests (acidfast bacilli stain, cultures on Lowenstein-Jensen media or GeneXpert PCR), more details aredescribed in Table 1. All clinical tests mentioned above were performed by the Hospital’sClinical Laboratory, which has a certified license issued by the National AIDS ReferenceLaboratory in China CDC. Treatment for the diagnosed HIV-TB patients consisted of standardfixed-dose chemotherapy for two weeks with isoniazid (H), ethambutol (E), rifampicin (R) andpyrazinamide (Z), followed by a combination of highly active antiretroviral therapy (HAART)and anti-TB treatment (HERZ) [1]. The HAART regimen consisted of Zidovudine plusLamivudine with Efavirenz, which is the recommended anti-HIV treatment regimen in China [2].All the patients were not treated with HAART before, and there was no evidence of co-infectionwith hepatitis B or C virus, cytomegalovirus, syphilis, toxoplasma. The criteria for TBassociatedIRIS was defined and classified according to the consensus adult TB-IRIS casedefinition developed by the International Network for Study of HIV-associated IRIS (INSHI) [3].More details are shown in Table 2. Blood samples (5 mL per patient/sample) from participants recruited at the Shenzhen ThirdPeople’s Hospital from April 2013 to September 2013 were collected. The plasma was test usingSemenAssay® Zinc Kit for determination of the Zinc Level. PBMCs were isolated immediatelyfrom the whole blood before, and two-weeks after the initiation of HAART. The total RNA wasextracted from the fresh PBMCs using the Qiagen RNeasy kit (QIAGEN). The mRNAs werepurified using oligo(dT) beads then used to construct RNA-seq libraries, which were sequencedon an Illumina HiSeq 2000 sequencing platform (using TruSeqV3 sequencing reagents ) at theBeijing Genomics Institute-Shenzhen, China (Expected library size: 200bp; Read length: 100nt;and Sequencing strategy: paired-end sequencing) as previously described [4].
Sample collection, PBMC RNA extraction and RNA-seq sequencing
All participants were screened for HIV antigens and antibodies via standard ELISA analyses,and these findings were further confirmed by Western Blotting. CD4+ T-cell counts wereobtained by flow cytometry, and the viral loads were measured by qRT-PCR. HIV serumpositivepatients were also diagnosed for M. tuberculosis, as assessed by microbiology tests (acidfast bacilli stain, cultures on Lowenstein-Jensen media or GeneXpert PCR), more details aredescribed in Table 1. All clinical tests mentioned above were performed by the Hospital’sClinical Laboratory, which has a certified license issued by the National AIDS ReferenceLaboratory in China CDC. Treatment for the diagnosed HIV-TB patients consisted of standardfixed-dose chemotherapy for two weeks with isoniazid (H), ethambutol (E), rifampicin (R) andpyrazinamide (Z), followed by a combination of highly active antiretroviral therapy (HAART)and anti-TB treatment (HERZ) [1]. The HAART regimen consisted of Zidovudine plusLamivudine with Efavirenz, which is the recommended anti-HIV treatment regimen in China [2].All the patients were not treated with HAART before, and there was no evidence of co-infectionwith hepatitis B or C virus, cytomegalovirus, syphilis, toxoplasma. The criteria for TBassociatedIRIS was defined and classified according to the consensus adult TB-IRIS casedefinition developed by the International Network for Study of HIV-associated IRIS (INSHI) [3].More details are shown in Table 2. Blood samples (5 mL per patient/sample) from participants recruited at the Shenzhen ThirdPeople’s Hospital from April 2013 to September 2013 were collected. The plasma was test usingSemenAssay® Zinc Kit for determination of the Zinc Level. PBMCs were isolated immediatelyfrom the whole blood before, and two-weeks after the initiation of HAART. The total RNA wasextracted from the fresh PBMCs using the Qiagen RNeasy kit (QIAGEN). The mRNAs werepurified using oligo(dT) beads then used to construct RNA-seq libraries, which were sequencedon an Illumina HiSeq 2000 sequencing platform (using TruSeqV3 sequencing reagents ) at theBeijing Genomics Institute-Shenzhen, China (Expected library size: 200bp; Read length: 100nt;and Sequencing strategy: paired-end sequencing) as previously described [4].
| Originalsprog | Engelsk |
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| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
|---|---|
| Status | Udgivet - 2018 |
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