Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR

Abstract

For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes. Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on K. pneumoniae. In this study, the expression profile of eleven candidate reference genes was assessed in K. pneumoniae cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta CT and RefFinder. The statistical analyses ranked recA, rho, proC and rpoD as the most suitable reference genes for accurate RT-qPCR data normalization in K. pneumoniae. The reliability of the proposed reference genes was validated by normalizing the relative expression of iron-regulated genes in K. pneumoniae cells submitted to iron-replete and iron-limited conditions. This work emphasizes that the stable expression of any potential reference candidate gene must be validated in each physiological condition or experimental treatment under study.

Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR

“…Inconsistencies in reported reference genes attributed to the lack of a universal reference gene for use in gene expression studies and, therefore, the expression stability of reference Selection and validation of reference genes for Pseudomonas brassicacearum GS20 using real-time-qPCR genes could vary under different experimental conditions [20]. Ideally, the expression stability of each candidate reference gene should be verified under each condition [29]. Other studies used 16S rRNA genes as internal references for validation [30,31], but transcript levels of 16S may depend on the state of the bacteria [32].…”

cis-9-Hexadecenal, a Natural Compound Targeting Cell Wall Organization, Critical Growth Factor, and Virulence of Aspergillus fumigatus

“…36 Reference genes are "house-keeping" genes whose expression remains constant at variant physiological conditions and are used for gene normalization. 36 In the present study, β-actin was used as reference gene and expression stability was analyzed on the basis of threshold cycle (ΔC t ) value. 36 The relative expression of pksP/alb1 gene was 3.5-fold (p ≤ 0.0005) upregulated in C-9-H-treated sample as compared to WT sample, whereas there was no expression of pksP/alb1 gene in ΔpksP A. fumigatus (Figure 7).…”

“…36 In the present study, β-actin was used as reference gene and expression stability was analyzed on the basis of threshold cycle (ΔC t ) value. 36 The relative expression of pksP/alb1 gene was 3.5-fold (p ≤ 0.0005) upregulated in C-9-H-treated sample as compared to WT sample, whereas there was no expression of pksP/alb1 gene in ΔpksP A. fumigatus (Figure 7). Upregulation of the gene expression is attributed in response to the development of any stress in the pathogen.…”

“…qRT-PCR amplification was carried out by using HiMedia LA-1012 Insta Q96-Real Time PCR, using a SYBR-green master mix (HiMedia, India), and the relative quantification of each individual expression of pksP/alb1 gene was performed using the comparative threshold cycle method. 36 The amplification program used for real time was 95°C for 3 min, 40 A. fumigatus wet mat (1.5 g) with or without any treatment was ground in liquid nitrogen separately, and total protein was isolated at 4°C in chilled 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM EDTA (HiMedia, India), 0.2 mM dithiothreitol (HiMedia, India), and 1 mM PMSF (HiMedia, India), for 3 h under constant stirring. The extracted protein was then centrifuged at 12 000 rpm for 20 min at 4°C.…”

Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

“…Reactions were analyzed using StepOnePlus Real Time PCR system (ThermoFisher Scientific). All reactions were performed in three biological replicates and two technical replicates, and expression was normalized to the reference genes, recA and rho 25 .…”