Validation of Stable Housekeeping Genes for Quantitative Real Time PCR in Golden Syrian Hamster

2020 ◽  
Author(s):  
Jin-xin Miao ◽  
Jian-yao Wang ◽  
Nick Robl ◽  
Hao-ran Guo ◽  
Shao-he Song ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Real Time ◽  
Syrian Hamster ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Pancreatic Tissue ◽  
Golden Syrian Hamster ◽  
The Stability ◽  
Selection Of

Background: Golden Syrian hamster (GSH) have many advantages as animal models in preclinical research, but their application is currently limited by the lack of standardized techniques for analyzing gene expression. Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for analyzing gene expression, but its reliability depends upon the selection of stable reference (“housekeeping”) genes for proper normalization. The current study was aimed to RT-qPCR for investigated to determine the stability housekeeping gene in golden Syrian hamster.Methods: During the period of June 2019 to October 2019, the expression stability of eight commonly-used housekeeping genes (glyceraldehyde-3phosphate dehydrogenase, Gapdh; b-actin, Actb; hypoxanthine phosphoribosyl transferase 1, Hprt1; ribosomal protein L13a, Rpl13a; ribosomal protein S18, Rps18; Beta-2-microglobulin, B2m; Tubulin beta class I, Tubb; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta, Ywhaz) were investigated in lung, liver, spleen and pancreatic tissue of golden Syrian hamsters using BestKeeper, geNorm, NormFinder software, comparative delta Ct method and RefFinder. Result: It was found that the stability of gene expression varied among tissue where in Actb was the most stably expressed housekeeping gene for lung tissue, Hprt1 for liver, Rpl13a for spleen and Tubb for pancreatic tissue. In contrast, the least stable housekeeping gene in both liver and spleen was Gapdh, Rps18 in pancreas and Ywhaz in lung tissue. These data provide a critical evaluation of housekeeping genes in GSH tissues that can be used as a guide for selection of the appropriate genes in future studies.

scholarly journals Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury

2019 ◽  
Vol 10 (1) ◽  
pp. 6
Author(s):  
Ján Košuth ◽  
Martina Farkašovská ◽  
Filip Mochnacký ◽  
Zuzana Daxnerová ◽  
Juraj Ševc
Keyword(s):  
Gene Expression ◽  
Spinal Cord ◽  
Spinal Cord Injury ◽  
Postnatal Development ◽  
Cell Metabolism ◽  
Housekeeping Genes ◽  
Protein Translation ◽  
Cord Injury ◽  
The Stability ◽  
Selection Of

In order to obtain unbiased results of target gene expression, selection of the most appropriate reference gene (RG) remains a key precondition. However, an experimental study focused on the validation of stably expressed RGs in the rat spinal cord (SC) during development or after spinal cord injury (SCI) is missing. In our study, we tested the stability of the expression of nine selected RGs in rat SC tissue during normal development (postnatal days 1–43, adulthood) and after minimal (mSCI) and contusion (cSCI) spinal cord injury. The following RGs were tested: common housekeeping genes of basal cell metabolism (Gapdh, Hprt1, Mapk6) and protein translation (Rpl29, Eef1a1, Eif2b2), as well as newly designed RGs (Gpatch1, Gorasp1, Cds2) selected according to the RefGenes tool of GeneVestigator. The stability of RGs was assessed by geNorm, NormFinder, and BestKeeper. All three applets favored Gapdh and Eef1a1 as the most stable genes in SC during development. In both models of SCI, Eif2b2 displayed the highest stability of expression, followed by Gapdh and Gorasp1/Hprt1 in cSCI, and Gapdh and Eef1a1 in the mSCI experiments. To verify our results, selected RGs were employed for normalization of the expression of genes with a clear biological context in the SC—Gfap and Slc1a3/Glast during postnatal development and Aif1/Iba1 and Cd68/Ed1 after SCI.

194 HOUSEKEEPING GENE EXPRESSION OF BOVINE FERTILIZED AND CLONED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 195 ◽  
Author(s):  
P. J. Ross ◽  
K. Wang ◽  
Z. Beyhan ◽  
A. Kocabas ◽  
J. B. Cibelli
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
18S Rrna ◽  
Cyclophilin A ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Blastocyst Stage ◽  
Histone H2a ◽  
Cell Stage ◽  
Expression Levels

Real-time RT-PCR can accurately quantify mRNA levels in pre-implantation embryos; however, comparisons among different embryonic stages and among embryos produced by different means often rely on a control gene, which is commonly assumed to remain constant across samples. The objective of this study was to compare housekeeping gene expression levels, relative to total mRNA, across different stages of bovine pre-implantation development in embryos generated by IVF and somatic cell nuclear transfer (SCNT). Embryos were produced according to standard protocols (Ross et al. 2006 Biotechniques 41, 741–750). Total RNA was collected from 3 pools of 10 oocyte/embryos at metaphase II (MII), PN, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages and reverse-transcribed using oligo-dT primers. The cDNA was then amplified using PCR (Kocabas et al. 2006 PNAS 103, 14 027–14 032). All amplified cDNA samples were diluted to 1 ng μL–1, as determined by NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and corroborated using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). For real-time PCR, 2 μL of cDNA was analyzed in duplicates. Absolute quantification was performed as previously described (Iager et al. 2008 Cloning Stem Cells doi:10.1089), using SYBR-green chemistry and standard curves specific for each gene. The number of RNA copies per nanogram of amplified cDNA was compared among samples using ANOVA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin A, β-actin, ribosomal protein L-15 (RPL-15), and β-glucuronidase (GUS) expression levels were similar in MII oocyte, 1-, 2-, 4-, and 8-cell embryos, while a significant increase at morula and blastocyst stages was observed (P < 0.05). A similar pattern of expression was observed for 18S ribosomal RNA, but with a significant decrease from morula to blastocyst stages (P < 0.05). Histone H2A expression was significantly higher at 1-cell stage, similar from 2-cell to morula stages and lowest at the blastocyst stage. We then compared expression between IVF and SCNT embryos at 2-, 4-, and 8-cell and blastocyst stages. GAPDH, RPL-15, GUS, and β-actin were significantly different among groups in at least 3 of the analyzed stages, which in all cases included blastocysts. 18s-rRNA was different among IVF and SCNT embryos only at the 8-cell stage, while no differences were observed at any stages for histone H2A and cyclophilin A. At the blastocyst stage, the lowest overall variability among IVF and SCNT embryos was observed for 18s-rRNA. In conclusion, none of the evaluated housekeeping genes showed consistent mRNA expression levels across developmental stages of IVF embryos. In addition, SCNT embryos, compared to IVF, had different levels of gene expression for commonly used housekeeping genes, which, if neglected, might result in data misinterpretation. In our conditions, histone H2A had similar expression levels between IVF and SCNT embryos across different stages and showed less variability than cyclophilin A. Finally, for comparisons at the blastocyst stage, 18s-rRNA had the least variability among IVF and SCNT embryos.

Selection of reference genes for quantitative real-time RT-PCR on gene expression in Golden Pompano (Trachinotus ovatus)

2017 ◽  
Vol 20 (3) ◽  
pp. 583-594 ◽  
Author(s):  
X.J. Chen ◽  
X.Q. Zhang ◽  
S. Huang ◽  
Z.J. Cao ◽  
Q.W. Qin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Poly I:C ◽  
Stable Gene ◽  
Trachinotus Ovatus ◽  
Golden Pompano ◽  
Normal Physiological Condition ◽  
Selection Of

Abstract Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.

Selection of housekeeping genes for normalization by real-time RT–PCR: Analysis of Or-MYB1 gene expression in Orobanche ramosa development

2008 ◽  
Vol 379 (2) ◽  
pp. 176-181 ◽  
Author(s):  
C.I. González-Verdejo ◽  
J.V. Die ◽  
S. Nadal ◽  
A. Jiménez-Marín ◽  
M.T. Moreno ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Housekeeping Genes ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Orobanche Ramosa ◽  
Selection Of

scholarly journals Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR

2008 ◽  
Vol 9 (1) ◽  
pp. 28 ◽  
Author(s):  
Carlos Infante ◽  
Makoto P Matsuoka ◽  
Esther Asensio ◽  
José Cañavate ◽  
Michael Reith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Genes ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Selection Of

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

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Ren Chen ◽  
Mayumi Gyokusen ◽  
Yoshihisa Nakazawa ◽  
Koichiro Gyokusen
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Transgene Expression ◽  
Pcr Amplification ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Amplification Efficiency ◽  
Eucommia Ulmoides ◽  
Rt Pcr

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.

Validation of housekeeping genes for studying differential gene expression in the bovine myometrium

2013 ◽  
Vol 61 (4) ◽  
pp. 505-516 ◽  
Author(s):  
Robert Rekawiecki ◽  
Magdalena Kowalik ◽  
Jan Kotwica
Keyword(s):  
Gene Expression ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Receptor Expression ◽  
Physiological Status ◽  
Stability Factor ◽  
Progesterone Receptor Expression ◽  
Expression Stability ◽  
Differential Gene ◽  
The Stability

The aim of this study was to determine the steady-state expression of 13 selected housekeeping genes in the myometrium of cyclic and pregnant cows. Cells taken from bovine myometrium on days 1–5, 6–10, 11–16 and 17–20 of the oestrous cycle and in weeks 3–5, 6–8 and 9–12 of pregnancy were used. Reverse transcribed RNA was amplified in real-time PCR using designed primers. Reaction efficiency was determined with the Linreg programme. The geNorm and NormFinder programmes were used to select the best housekeeping genes. They calculate the expression stability factor for each used housekeeping gene with the smallest value for most stably expressed genes. According to geNorm, the most stable housekeeping genes in the myometrium were C2orf29, TPB and TUBB2B, while the least stably expressed genes were 18S RNA, HPRT1 and GAPDH. NormFinder identified the best genes in the myometrium as C2orf29, MRPL12 and TBP, while the worst genes were 18S RNA, B2M and SF3A1. Differences in stability factors between the two programmes may also indicate that the physiological status of the female, e.g. pregnancy, affects the stability of expression of housekeeping genes. The different expression stability of housekeeping genes did not affect progesterone receptor expression but it could be important if small differences in gene expression were measured between studies.

scholarly journals Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis of the veined rapa whelk (Rapana venosa)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3398 ◽  
Author(s):  
Hao Song ◽  
Xin Dang ◽  
Yuan-qiu He ◽  
Tao Zhang ◽  
Hai-yan Wang
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Developmental Stages ◽  
Housekeeping Genes ◽  
Internal Controls ◽  
Rapana Venosa ◽  
Qrt Pcr ◽  
Different Tissues ◽  
Selection Of ◽  
Rapa Whelk

BackgroundThe veined rapa whelkRapana venosais an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression inR. venosa. For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes inR. venosafor use as internal controls for qRT-PCR.MethodsIn this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1α(EF-1α),α-actin (ACT), cytochrome c oxidase subunit 1 (COX1), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1αsubcomplex subunit 7 (NDUFA7), 60S ribosomal protein L5 (RL5), 60S ribosomal protein L28 (RL28), glyceraldehyde 3-phosphate dehydrogenase (GAPDH),β-tubulin (TUBB), 40S ribosomal protein S25 (RS25), 40S ribosomal protein S8 (RS8), ubiquitin-conjugating enzyme E2 (UBE2), histone H3 (HH3), and peptidyl-prolyl cis-trans isomerase A (PPIA). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.ResultsOf the 13 candidate genes tested, we found thatEF-1αwas the most stable internal control gene in almost all adult tissue samples investigated withRL5andRL28as secondary choices. For the normalization of a single specific tissue, we suggested thatEF-1αandNDUFA7are the best combination in gonad, as well asCOX1andRL28for intestine,EF-1αandRL5for kidney,EF-1αandCOX1for gill,EF-1αandRL28for Leiblein and mantle,EF-1α,RL5, andNDUFA7for liver, GAPDH,PPIA, andRL28for hemocyte. From a developmental perspective, we found thatRL28was the most stable gene in all developmental stages measured, andCOX1andRL5were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization,PPIA,RS25, andRL28for stage 1,RL5andRL28for stage 2 and 5,RL28andNDUFA7for stage 3, andPPIAandTUBBfor stage 4.DiscussionOur results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development ofR. venosain the future.

RPS24c Isoform Facilitates Tumor Angiogenesis Via Promoting the Stability of MVIH in Colorectal Cancer

2020 ◽  
Vol 20 (5) ◽  
pp. 388-395 ◽  
Author(s):  
Yue Wang ◽  
Youjun Wu ◽  
Kun Xiao ◽  
Yingjie Zhao ◽  
Gang Lv ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Ribosomal Protein ◽  
Real Time ◽  
Tumor Angiogenesis ◽  
Real Time Pcr ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Binding Interaction ◽  
Tubule Formation ◽  
The Stability

Background: Colorectal cancer (CRC) is the second leading cause of death worldwide, and distant metastasis is responsible for the poor prognosis in patients with advanced-stage CRC. RPS24 (ribosomal protein S24) as a ribosomal protein, multiple transcript variant encoding different isoforms have been found for this gene. Our previous studies have demonstrated that RPS24 is overexpressed in CRC. However, the mechanisms underlying the role of RPS24 in tumor development have not been fully defined. Methods: Expression of RPS24 isoforms and lncRNA MVIH in CRC tissues and cell lines were quantified by real-time PCR or western blotting assay. Endothelial tube formation assay was performed to determine the effect of RPS24 on tumor angiogenesis. The cell viability of HUVEC was determined by MTT assay, and the migration and invasion ability of HUVEC were detected by transwell assay. PGK1 secretion was tested with a specific ELISA kit. Results: Here, we found that RPS24c isoform was a major contributor to tumor angiogenesis, a vital process in tumor growth and metastasis. Real-time PCR revealed that RPS24c isoform was highly expressed in CRC tissues, while other isoforms are present in both normal and CRC tissues with no statistical difference. Moreover the change of RPS24 protein level is mainly due to the fluctuation of RPS24c. Furthermore, we observed that silencing RPS24c could decrease angiogenesis by inhibiting tubule formation, HUVEC cell proliferation and migration. Additionally, we investigated the molecular mechanisms and demonstrated that RPS24c mRNA interacted with lncRNA MVIH, the binding-interaction enhanced the stability of each other, thereby activated angiogenesis by inhibiting the secretion of PGK1. Conclusion: RPS24c facilitates tumor angiogenesis via the RPS24c/MVIH/PGK1 pathway in CRC. RPS24c inhibition may be a novel option for anti-vascular treatment in CRC.

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