Peer Review #2 of "Determination of the reference genes for qRT-PCR normalization and expression levels of MAT genes under various conditions in Ulocladium (v0.1)"

Abstract The quantitative real-time transcription-polymerase chain reaction (qRT-PCR) is now used widely in studies about mRNA expression levels. The selection of one or more stable reference gene(s) used for data normalization is substantial. In this study, expression levels of eleven candidate reference genes (β-actin, 16S rRNA, 18S rRNA, 28S rRNA, α-I tubulin, GAPDH, ribosomal protein L13, elongation factor 1 α, elongation factor 2, arginine kinase and ubiquitin) were examined using the GenomeLab GeXP analysis system (Beckman Coulter). Gene expression data were analysed using two different statistical models: geNorm and NormFinder. (1) In six different tissues (hepatopancreas, haemocytes, heart, gill, muscle, and testis) from the mud crab, Scylla paramamosain, 18S rRNA and elongation factor 1 α were identified as the two best reference genes. (2) In the haemocytes after being challenged by Vibro parahaemolyticus, the result suggested that ubiquitin was the most stable gene after the treatment. 18S rRNA, elongation factor 1 α and ubiquitin are herein recommended as the best combination. These results provide useful options for reference gene selection under different experimental conditions in qRT-PCR studies in the mud crab.

Download Full-text

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Scientific Reports ◽

10.1038/s41598-021-81524-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Min-dong Chen ◽

Bin Wang ◽

Yong-ping Li ◽

Mei-juan Zeng ◽

Jian-ting Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Patterns ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference ◽

Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Download Full-text

Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Biology Methods and Protocols ◽

10.1093/biomethods/bpw005 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Roshini Kalagara ◽

Weimin Gao ◽

Honor L. Glenn ◽

Colleen Ziegler ◽

Laura Belmont ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Stable Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

Download Full-text

Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

Silvae Genetica ◽

10.1515/sg-2012-0035 ◽

2012 ◽

Vol 61 (1-6) ◽

pp. 280-286

Author(s):

B. Karpaga Raja Sundari ◽

M. Ghosh Dasgupta

Keyword(s):

Real Time ◽

Reference Genes ◽

Genomic Research ◽

Initiation Factor ◽

Primary Cell Wall ◽

Stable Gene ◽

Eucalyptus Tereticornis ◽

Expression Levels ◽

Qrt Pcr ◽

Different Tissues

AbstractReference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

Download Full-text

Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽

10.7717/peerj.6536 ◽

2019 ◽

Vol 7 ◽

pp. e6536 ◽

Cited By ~ 5

Author(s):

Li Miao ◽

Xing Qin ◽

Lihong Gao ◽

Qing Li ◽

Shuzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Stable Expression ◽

Quantitative Real Time Pcr ◽

Graft Union ◽

Expression Levels ◽

Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

Download Full-text