Identification and validation of Aeluropus littoralis reference genes for Quantitative Real-Time PCR Normalization

Abstract

BackgroundThe use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis—halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted.ResultsTen candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae.ConclusionsOverall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A.littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.Electronic supplementary materialThe online version of this article (doi:10.1186/s40709-016-0053-8) contains supplementary material, which is available to authorized users.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

“…However, gaining optimal benefits from these advantages requires a clear understanding of the setup of the parameters used for the qPCR experiment. To receive a reliable result in qPCR gene expression analysis, it is necessary to avoid the nonspecific amplification that arises from primer-dimer or gDNA contamination in the RNA sample 3 15 . It is expected that the RNA transcript levels will be overestimated under gDNA contamination 8 .…”

“…Since gDNA contamination is not distributed uniformly between different RNA samples, and the reaction sensitivity to gDNA is significantly affected by the genes analyzed, NRT controls are required for each sample/assay pair 7 15 . This will substantially add cost and labor when handling many samples simultaneously 3 9 . Other alternative methods documented in the literature include the use of intron specific primers for the detection of gDNA, or designing primers that either flank an intron or span an exon-exon junction.…”

“…Reverse transcription (RT) followed by qPCR is widely used for transcriptome analysis that measure gene expression levels in various areas of biological research 2 . Compared to other methods such as the traditional Northern hybridization, tissue specific detection via in situ hybridization, ribonuclease protection assays (RPA), and semi-RT-PCR, the accuracy, convenience, speed, and wide dynamic range of qPCR-based assays are highly remarkable 3 4 . There are several important factors that have to be considered for a reliable quantification of messenger RNA (mRNA), including the quality and quantity of RNA starting material.…”

“…Due to the high degree of repetitiveness, the ratio of genomic copy number and detectable unprocessed RNA premolecules is lower compared to the low-copy intron sequences and unspliced precursors. These features make rDNA genes well suited for reliable and highly sensitive detection of gDNA contamination in most eukaryotes and prokaryotes 3 .…”

Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut

“…Several researchers (Yael and Ran 2010, Morgante et al 2011, Reddy et al 2013 have reported different patterns of reference genes at different developmental stages, or various experimental conditions in peanut. Differential stability of reference genes in different tissues has also been reported in other plants (Lin et al 2013, Imai et al 2014, Hashemi et al 2016. Some reference genes, like ADH3 and TUA5, have previously been highlighted as the most stable genes in peanut, but they are not suitable to all experimental sets.…”