Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis inAphis gossypii(Hemiptera: Aphididae)

In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients–-a process that requires large sample sizes by combining independent sets of data.

Download Full-text

Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR

Journal of Fish Biology ◽

10.1111/jfb.12915 ◽

2016 ◽

Vol 88 (6) ◽

pp. 2095-2110 ◽

Cited By ~ 34

Author(s):

H. Xu ◽

C. Li ◽

Q. Zeng ◽

I. Agrawal ◽

X. Zhu ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Data ◽

Danio Rerio ◽

Reference Genes ◽

Expression Data ◽

Genome Wide

Download Full-text

Identification of stable reference genes in differentiating human pluripotent stem cells

Physiological Genomics ◽

10.1152/physiolgenomics.00130.2014 ◽

2015 ◽

Vol 47 (6) ◽

pp. 232-239 ◽

Cited By ~ 11

Author(s):

Gustav Holmgren ◽

Nidal Ghosheh ◽

Xianmin Zeng ◽

Yalda Bogestål ◽

Peter Sartipy ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Gene Expression Data ◽

Pluripotent Stem Cells ◽

Reference Genes ◽

Cell Types ◽

Human Pluripotent Stem Cells ◽

Expression Data ◽

Large Variability ◽

The Stability

Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs.

Download Full-text

Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

Author(s):

Jing-Jing Wang ◽

Shuo Han ◽

Weilun Yin ◽

Xinli Xia ◽

Chao Liu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

Pcr Analysis ◽

Qrt Pcr ◽

Gene Normalization ◽

Accurate Normalization ◽

Suitable Reference ◽

Different Tissues ◽

Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

Download Full-text

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

PLoS ONE ◽

10.1371/journal.pone.0169465 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169465 ◽

Cited By ~ 6

Author(s):

Dongli Wan ◽

Yongqing Wan ◽

Qi Yang ◽

Bo Zou ◽

Weibo Ren ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Environmental Stresses ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

Download Full-text

The normalization of gene expression data in melanoma: Investigating the use of glyceraldehyde 3-phosphate dehydrogenase and 18S ribosomal RNA as internal reference genes for quantitative real-time PCR

Analytical Biochemistry ◽

10.1016/j.ab.2008.05.024 ◽

2008 ◽

Vol 380 (1) ◽

pp. 137-139 ◽

Cited By ~ 26

Author(s):

Orsolya Giricz ◽

Janelle L. Lauer-Fields ◽

Gregg B. Fields

Keyword(s):

Gene Expression ◽

Real Time ◽

Gene Expression Data ◽

Ribosomal Rna ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Data ◽

Quantitative Real Time Pcr ◽

Internal Reference ◽

18S Ribosomal Rna

Download Full-text

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Chinese Journal of Oceanology and Limnology ◽

10.1007/s00343-015-4004-2 ◽

2014 ◽

Vol 32 (6) ◽

pp. 1248-1256 ◽

Cited By ~ 9

Author(s):

Ye Zhao ◽

Muyan Chen ◽

Tianming Wang ◽

Lina Sun ◽

Dongxue Xu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Sea Cucumber ◽

Apostichopus Japonicus ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

Download Full-text

Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

PeerJ ◽

10.7717/peerj.7133 ◽

2019 ◽

Vol 7 ◽

pp. e7133 ◽

Cited By ~ 4

Author(s):

Wen Zhou ◽

Shiqiang Wang ◽

Lei Yang ◽

Yan Sun ◽

Qian Zhang ◽

...

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Reference Genes ◽

Hypericum Perforatum ◽

Developmental Stages ◽

Pcr Analysis ◽

Potential Candidate ◽

Qrt Pcr ◽

Expression Studies ◽

Different Tissues

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.

Download Full-text