Robust Normalization of Luciferase Reporter Data.

Andrea Repele,Manu Manu

doi:10.3390/mps2030062

Abstract

Transient Luciferase reporter assays are widely used in the study of gene regulation and intracellular cell signaling. In order to control for sample-to-sample variation in luminescence arising from variability in transfection efficiency and other sources, an internal control reporter is co-transfected with the experimental reporter. The luminescence of the experimental reporter is normalized against the control by taking the ratio of the two. Here we show that this method of normalization, “ratiometric”, performs poorly when the transfection efficiency is low and leads to biased estimates of relative activity. We propose an alternative methodology based on linear regression that is much better suited for the normalization of reporter data, especially when transfection efficiency is low. We compare the ratiometric method against three regression methods on both simulated and empirical data. Our results suggest that robust errors-in-variables (REIV) regression performs the best in normalizing Luciferase reporter data. We have made the R code for Luciferase data normalization using REIV available on GitHub.

Highlights

Transient reporter assays are an important and widely used tool in the study of gene regulation [1,2,3,4], intracellular cell signaling [5,6,7], and other areas of molecular, cellular, and developmental biology [8,9,10]
A reporter of CCAAT/Enhancer binding protein, α (Cebpa) promoter expression exhibits ∼3-fold variation in luminescence when assayed in PUER cells [13,14,15], a myeloid cell line with low transfection efficiency (Figure 1; Section 3.1)
We evaluated the performance of four methods, ratiometric, Ordinary least-squares (OLS), EIV, and robust errors-in-variables (REIV), on simulated data, where the “ground truth” is known

Summary

Introduction

Transient reporter assays are an important and widely used tool in the study of gene regulation [1,2,3,4], intracellular cell signaling [5,6,7], and other areas of molecular, cellular, and developmental biology [8,9,10]. The activity of a promoter, potentially in combination with an enhancer, is measured by placing an inert reporter gene such as luciferase or lacZ under the control of the promoter. A reporter of CCAAT/Enhancer binding protein, α (Cebpa) promoter expression exhibits ∼3-fold variation in luminescence when assayed in PUER cells [13,14,15], a myeloid cell line with low transfection efficiency (Figure 1; Section 3.1). The prevalent method of correcting for sample-to-sample luminescence variation [3,8] is to co-transfect an independent control reporter, such as Renilla luciferase expressed from a constitutive promoter, along with the promoter/enhancer reporter being assayed. Since transfection efficiency and other experimental errors would affect both constructs the activity of the experimental promoter relative to the constitutive promoter can be determined by taking the ratio of firefly and Renilla luminescence.

Objectives

Methods

Results