Repeatability and reproducibility of an automated, multiplex p450 enzyme gene expression assay JasonDeLoach1, Dominic LaRoche1, Qian Liu1, Byron Lawson1, Janet Rothers2 and MarkSchwartz1

1HTG Molecular Diagnostics, Inc., 2Consultant

Rationale and Objective Methods Results

HTG Edge system & chemistry

HTG Edge system

HTG Edge chemistry

HTG Molecular Diagnostics | 3430 E. Global Loop | Tucson, AZ 85706 | (877) 289-2615 | htgmolecular.com | Presented at 20th North American ISSX Meeting

For Research Use Only. Not for use in diagnostic procedures.

HTG EdgeSeq, HTG Edge and qNPA are trademarks of HTG Molecular Diagnostics, Inc. Any other trademarks or trade names used herein are the intellectual property of their respective owners.

Table 2. Correlations Between All Pair-Wise Replicates By Day

Table 1. Correlations Between All Pair-Wise Replicates By Processor

MaterialsPrimary human hepatocytes (donor lot DQB) were sourced from BioreclamationIVT.

InductionPrototypical inducers and control were used to treat hepatocytes:

- Vehicle Control (DMSO): 0.1% DMSO - Omeprazole (OME): 50 µM- Phenobarbital (PB): 1000 µM- Rifampicin (RIF): 50 µM

Media removed and cells lysed in HTG Lysis Buffer after 48 hours

HTG Edge system Repeatability and Reproducibility StudiesEquivalents of 25,000, 12,500, 6,250 and 3,125 lysed cells per well were prepared (triplicate wells for each treatment) using 96 well HTG Edge sample plates according to the following layout:

Repeatability: 3 runs on 3 different days on single HTG Edge processor Reproducibility: 3 runs on 3 separate HTG Edge processors

HTG Edge DMPK Core AssayGene expression from 3 p450 enzymes measured (CYPs: 1A2, 2B6 and 3A4) Housekeeping genes: GAPDH, EEF2

, ACTB, RPLP0Controls: ANT, POS1

Data AnalysisAnalyses are primarily qualitative and conditional on three assays: HTG Edge DMPK Comprehensive, HTG Edge DMPK Core, and HTG Edge DMPK Core HB; as well as the induction treatments and vehicle control described above.

Overall expression is displayed in the form of boxplots. Horizontal lines denote the median expression values, and the edges of the boxes denote the 25th and 95th

percentiles, respectively. Vertical lines extend to most extreme observations, or end at 1.95 times the inter-quartile range, with observations that exceed this value represented by dots.

Correlations between conditions were calculated by first averaging expression between triplicate samples within each processor and day condition. Total number of technical replicates was 8. Technical replicates are expected to achieve a Pearson correlation > 0.90 between all technical replicates by condition.

Finally, we evaluated repeatability and reproducibility for the log2 fold-changes in each treatment to the vehicle control by comparing means and 95% confidence intervals (constructed based on the large sample assumption of normality around the ratio of normalized expression values).

Drug metabolizing enzymes and transporter induction can result in clinically meaningful drug interactions. It is therefore important to identify potential drug interactions early in the drug development process. Both p450 gene and enzyme induction studies are helpful in identifying p450 inducing compounds. Measuring gene expression has traditionally relied upon RNA extraction from treated hepatocytes followed by RT-qPCR. An alternative, potentially more efficient method for measuring gene induction in this setting is the automated multiplex HTG Edge system and HTG Edge chemistry, quantitative nuclease protection assay (qNPA).

We evaluated HTG Edge system’s multiplex p450 enzyme gene expression assay performance by assessing repeatability across multiple days and reproducibility across multiple instruments.

Distribution of all probe expression by day

Mean and 95% confidence intervals for fold-changesbetween treatment and vehicle control

Distribution of all probe expression by processor

Overall expression across conditions Distributions of all probe expression were similar across 3 different processors and 3 different days, as demonstrated by the boxplots below.

Fold change comparison across conditionsA comparison of means and 95% confidence for fold change across conditions demonstrates a high degree of precisionwithin treatments of each p450 enzyme.

Correlation across conditionsPearson correlation coefficients from pairwise comparisons between processors and days were greater than or equal to 0.90 for all treatments. The majority of correlation coefficients exceeded 0.95.

ConclusionThese analyses demonstrate the reproducibility and repeatability of HTG Edge system’s multiplex p450 enzyme gene expression assay for three separate induction treatments, across 3 different processors and across 3 different days.

1 2 3 4 5 6 7 8 9 10 11 12A 0.1% DMSO - Replicate 1 20uM RIF - Replicate 4 750uM PB - Replicate 6 50uM OME - Replicate 8B 50uM OME - Replicate 1 0.1% DMSO - Replicate 3 20uM RIF - Replicate 6 750uM PB - Replicate 8C 750uM PB - Replicate 1 50uM OME - Replicate 3 0.1% DMSO - Replicate 5 20uM RIF - Replicate 8D 20uM RIF - Replicate 1 750uM PB - Replicate 3 50uM OME - Replicate 5 0.1% DMSO - Replicate 7E 0.1% DMSO - Replicate 2 20uM RIF - Replicate 3 750uM PB - Replicate 5 50uM OME - Replicate 7F 50uM OME - Replicate 2 0.1% DMSO - Replicate 4 20uM RIF - Replicate 5 750uM PB - Replicate 7G 750uM PB - Replicate 2 50uM OME - Replicate 4 0.1% DMSO - Replicate 6 20uM RIF - Replicate 7H 20uM RIF - Replicate 2 750uM PB - Replicate 4 50uM OME - Replicate 6 0.1% DMSO - Replicate 8

Assay Treatment Proc+23+vs+31 Proc+28+vs+23 Proc+28+vs+31

ComprehensiveDMSO 0.93 0.96 0.93OME 0.95 0.98 0.95PB 0.94 0.95 0.94RIF 0.93 0.97 0.91

CoreDMSO 0.98 0.98 0.98OME 1.00 0.99 1.00PB 1.00 0.99 0.99RIF 1.00 0.98 0.99

Assay Treatment Proc+23+vs+82 Proc+28+vs+23 Proc+28+vs+82

HBDMSO 0.96 0.95 0.94OME 0.99 0.98 0.99PB 0.95 0.96 0.96RIF 0.95 0.97 0.93

Assay Treatment Day*1*vs*2 Day*1*vs*3 Day*2*vs*3

Comprehensive

DMSO 0.98 0.96 0.97OME 0.98 0.98 0.98PB 0.99 0.98 0.97RIF 0.97 0.97 0.97

Core

DMSO 0.98 0.99 0.97OME 1.00 1.00 0.99PB 0.99 1.00 0.99RIF 0.99 1.00 0.99

HB

DMSO 0.96 0.90 0.91OME 0.98 0.99 0.98PB 0.94 0.97 0.95RIF 0.95 0.98 0.97

Sample to answer in 24-28 hours

Run samples on HTG Edge processor22-27 hours

Sample Prep/Order Entry30 minutes

Transfer samples/run on HTG Edge reader40 minutes

Results/Reports5 minutesFSD JMJLLK

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