Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

Abstract

In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT-) PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT-) PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

Analysis of DNA Methylation Patterns Associated with In Vitro Propagated Globe Artichoke Plants Using an EpiRADseq-Based Approach

“…cDNA of true-to-type and off-type samples were tested on Step One Plus Real Time PCR (Applied Biosystem) and the transcript level was calculated using the ΔΔCT method. As housekeeping genes, actin (XM_025103545) and elongation factor 1 (EF1, XM_025112661) were chosen for their stability of transcript level [53]. RT-qPCRs were performed in three biological and three technical replicates using 2X Power SYBR ® Green PCR Master Mix (Applied Biosystem).…”

Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity *

“…Samples from each strain were run with five replicates. The rbcL gene was used as a reference (36). Expression levels were calculated with the CFX manager software (Bio-Rad) using the ⌬⌬Ct method.…”

Micro real-time PCR device using a circulation pump

“…8b). The efficiency of the DNA amplification was estimated to be 113 %, in which the reasonable range is known to be between 80 and 120 % (Chen et al 2010). The reason why the efficiency is larger than 100 % could be explained as follows.…”