Normalization of cell associated antiretroviral drug concentrations with a novel RPP30 droplet digital PCR assay

Scientific Reports
Shetty Ravi DyavarAnthony T Podany

Abstract

Quantification of antiretroviral (ARV) drug concentrations in peripheral blood mononuclear cells (PBMCs) and tissue isolated mononuclear cells (TIMCs) from lymph node (LNMC) and rectum (RMC) is an important measure of bio-distribution. Normalization of drug concentrations is critical to represent tissue drug concentrations and to analyze both intra-individual and inter-individual variability in drug distribution. However, a molecular method to normalize intracellular drug concentrations in PBMCs and TIMCs methanol extracts is currently unavailable. In this study, a novel droplet digital PCR (ddPCR) assay was designed to amplify RPP30 gene sequence conserved in human and non-human primates (NHP). Genomic DNA (gDNA) isolated from 70 percent methanol embedded PBMCs and TIMCs was used as ddPCR template to quantitate precise RPP30 copies to derive cell counts. The novel molecular method quantitated RPP30 copies in human and rhesus macaque gDNA templates with greater accuracy and precision than qPCR. RPP30 ddPCR derived cell counts are strongly correlated with automated cytometer based cell counts in PBMC (R = 0.90, p = 0.001 and n = 20); LNMC (R = 0.85 p = 0.0001 and n = 22) and RMC (R = 0.92, p = 0.0001 and n = 20) and achieved com...Continue Reading

References

Mar 1, 1990·Journal of Clinical Microbiology·R BoomJ van der Noordaa
Feb 25, 1990·Nucleic Acids Research·J D ThompsonD Gillepsie
Aug 10, 2005·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·Adrienne L McNeesJanet S Butel
Jul 11, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Tracy KingCourtney V Fletcher
Feb 27, 2007·Microscopy Research and Technique·Mirella FalconiGiovanni Mazzotti
Mar 12, 2009·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Tony K F YungY M Dennis Lo
Jun 6, 2009·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Tom DelahuntyCourtney V Fletcher
Nov 1, 2011·Analytical Chemistry·Benjamin J HindsonBill W Colston
Apr 24, 2012·The Journal of Clinical Investigation·Ravi Dyavar ShettyRama Rao Amara
May 3, 2013·Journal of Clinical Microbiology·Chrissy H RobertsMartin J Holland
Sep 3, 2013·Nature Methods·Christopher M HindsonMuneesh Tewari
Jan 29, 2014·Proceedings of the National Academy of Sciences of the United States of America·Courtney V FletcherTimothy W Schacker
Apr 26, 2014·Bioinformatics·Matthew N McCallAnthony Almudevar
Feb 26, 2015·Proceedings of the National Academy of Sciences of the United States of America·Meghan K RothenbergerTimothy W Schacker
Jan 28, 2016·Nature·Ramon Lorenzo-RedondoSteven M Wolinsky
Apr 18, 2015·Biotechnology Reports·Daniela Cadena-HerreraEmilio Medina-Rivero

❮ Previous
Next ❯

Citations

Jul 25, 2019·The Journal of Antimicrobial Chemotherapy·Shetty Ravi DyavarCourtney V Fletcher
Feb 5, 2021·Journal of Medical Virology·Amir Asri KojabadMajid Mirzaei Nodooshan

❮ Previous
Next ❯

Methods Mentioned

BETA
flow cytometry
PCR
electrophoresis
xenograft
biopsies
chip
chips

Software Mentioned

Biorad Quantity One 1 - D Analysis
Quantasoft Pro
Graph Pad
Prism
Quant studio ( QS )
ddPCR
Clustal Omega
QS3D
EVOS

Related Concepts

Related Feeds

Autoimmune Diseases

Autoimmune diseases occur as a result of an attack by the immune system on the body’s own tissues resulting in damage and dysfunction. There are different types of autoimmune diseases, in which there is a complex and unknown interaction between genetics and the environment. Discover the latest research on autoimmune diseases here.

Related Papers

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
Ping ZhangXiao-ning Xu
Journal of Biological Regulators and Homeostatic Agents
D CiuffredaF Lillo
© 2022 Meta ULC. All rights reserved