scholarly journals TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

2020 ◽  
Vol 33 (12) ◽  
pp. 2021-2030
Author(s):  
Si-Jung Jang ◽  
Ryoung-Hoon Jeon ◽  
Hwan-Deuk Kim ◽  
Jong-Chan Hwang ◽  
Hyeon-Jeong Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Ribosomal Protein ◽  
Reference Genes ◽  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Tata Box ◽  
Chain Reaction ◽  
Experimental Conditions ◽  
Polymerase Chain ◽  
Tata Box Binding Protein

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs.Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins.Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance.Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos

2008 ◽  
Vol 20 (4) ◽  
pp. 513 ◽  
Author(s):  
Bartosz Kempisty ◽  
Paweł Antosik ◽  
Dorota Bukowska ◽  
Marta Jackowska ◽  
Margarita Lianeri ◽  
...  
Keyword(s):  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Analysis ◽  
Tata Box ◽  
Chain Reaction ◽  
Camp Response Element ◽  
Cell Stage ◽  
Polymerase Chain ◽  
Tata Box Binding Protein ◽  
Porcine Spermatozoa

It has been suggested that spermatozoa can deliver mRNAs to the oocyte during fertilisation. Using reverse transcription and real-time quantitative polymerase chain reaction analysis (RQ-PCR), we evaluated the presence of clusterin (CLU), protamine 2 (PRM2), calmegin (CLGN), cAMP-response element modulator protein (CREM), methyltransferase 1 (DNMT1), linker histone 1 (H1), protamine 1 (PRM1), TATA box-binding protein associated factor 1 (TAF1) and TATA box-binding protein (TBP) in porcine mature oocytes, zygotes and two-cell stage embryos. Spermatozoa isolated from semen samples of boars contained all transcripts investigated, whereas oocytes contained only CREM, H1, TAF1, and TBP mRNAs. The zygote and two-cell stage embryos contained CLU, CREM, H1, PRM1, PRM2, TAF1 and TBP transcripts. Our observations suggest that porcine spermatozoa may delivery CLU, PRM1 and PRM2 mRNAs to the oocyte, which may contribute to zygotic and early embryonic development.

scholarly journals Validation of reference genes for accurate normalization by quantitative polymerase chain reaction in sugarcane drought stress studies using two cultivars

2018 ◽  
Vol 48 (11) ◽  
Author(s):  
Dennis Crystian ◽  
Jackeline Terto ◽  
José Vieira Silva ◽  
Cícero Almeida
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Drought Stress ◽  
Reference Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Elongation Factor ◽  
Chain Reaction ◽  
Stress Studies ◽  
Accurate Normalization ◽  
Polymerase Chain

ABSTRACT: The aim of this study was to analyze the expression of putative reference genes in sugarcane under drought stress. The varieties RB72454 and RB72910 were cultivated and the treatments control and drought stress applied to 135-day-old plants grown under field conditions. The stress level of the plants was measured by rate of photosynthesis, transpiration, and stomatal conductance. For each biological replicate, expression analyses were conducted using quantitative polymerase chain reaction for the genes α-tubulin, β-tubulin, β-actin, cyclophilin, eukaryotic elongation factor 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H3 and ubiquitin. Stability was evaluated using the geNorm, NormFinder, and BestKeeper software packages. Among the candidate genes, GAPDH was identified as the most stable in all software, indicating its suitability for gene expression studies in sugarcane undergoing drought stress; the gene β-actin was the second most stable. These findings suggest using GAPDH and β-actin for normalization in relative gene expression in sugarcane.

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