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

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 comparable normalized drug concentrations. Therefore, the RPP30 ddPCR assay is an important normalization method in drug bio-distribution and pharmacokinetic studies in humans and NHPs.

RPP30, a transcriptional regulator, is a potential pathogenic factor in glioblastoma

“…Further, we explored the specific modification functions of RPP30 at each stage of gene expression. Consistent with previous research findings, we found that RPP30 was involved in the modification of RNA, not DNA, in both tumor and non-tumor samples [ 14 , 15 , 18 ]. Surprisingly, we found that RPP30 was closely related to protein modification in non-tumor brain samples, but not in primary GBM samples in both the CGGA and TCGA databases ( Figure 2C ).…”

“…RNase P is also known as RNA polymerase III and is one of three major nuclear RNA polymerases in human cells. Studies have shown that in addition to the modification of tRNA, RNase P is involved in the regulation of gene transcription and cell cycle [ 14 , 15 ]. Previous work has reported that RPP30 plays a role in tumorigenesis and malignant progression in breast, ovarian, and lung cancers [ 16 – 18 ].…”

Hepatocytic transcriptional signatures predict comparative drug interaction potential of rifamycin antibiotics

“…PHHs were treated with rifampin, rifabutin and rifapentine at 10 µM concentration for 72 h. with the corresponding untreated (negative) and 0.0025% methanol (vehicle) treated controls. We measured intracellular concentrations (ICC) of parent and des-acetyl-metabolites by liquid chromatography tandem mass spectroscopy (LC-MS/MS) as we have previously described [45][46][47][48][49] .…”

Transcriptomic approach predicts a major role for transforming growth factor beta type 1 pathway in L‐Dopa‐induced dyskinesia in parkinsonian rats

“…After 40 cycles, an extension step was performed at 72°C for 10 minute and a final step of DNA polymerase deactivation step at 95°C for 5 min. FC in mRNA expression level of each sample was determined using ΔΔCT method 74 and GAPDH was used as internal control in qPCR assays. Three minimum replicates were used for all samples in qPCR assays (n = 3).…”