Evaluation of internal reference genes in Auxenochlorella protothecoides under continuous heterotrophic culture conditions at normal, low and high temperatures

2020 ◽  
Vol 49 ◽  
pp. 101941
Author(s):  
GuanLan Xing ◽  
Kui Liu ◽  
WenLi Li ◽  
JinYu Li ◽  
Chao Xing ◽  
...  
Keyword(s):  
High Temperatures ◽  
Reference Genes ◽  
Culture Conditions ◽  
Heterotrophic Culture ◽  
Internal Reference ◽  
Auxenochlorella Protothecoides

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

2012 ◽  
Vol 44 (12) ◽  
pp. 651-656 ◽  
Author(s):  
S. Ellefsen ◽  
M. Bliksøen ◽  
A. Rutkovskiy ◽  
I. B. Johansen ◽  
M.-L. Kaljusto ◽  
...  
Keyword(s):  
Infarct Size ◽  
Real Time ◽  
Reference Genes ◽  
Stable Expression ◽  
Unit Weight ◽  
Living Tissue ◽  
Rt Pcr ◽  
Internal Reference ◽  
Total Rna ◽  
Rat Hearts

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1–50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

scholarly journals Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marcelle SanJuan Ganem Prado ◽  
Thaline Cunha de Goes ◽  
Mirthz Lemos de Jesus ◽  
Lucilla Silva Oliveira Mendonça ◽  
Jadson Santos Nascimento ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Mirna Expression ◽  
Reference Genes ◽  
Data Interpretation ◽  
Stable Gene ◽  
Internal Reference ◽  
Expression Levels ◽  
Rna Molecules ◽  
Gene Normalization ◽  
Mirna Expression Data

AbstractDiabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.

Photosynthetic growth of tobacco cells in liquid suspension

1972 ◽  
Vol 50 (11) ◽  
pp. 2265-2270 ◽  
Author(s):  
M. Trevor Chandler ◽  
Nicole Tandeau de Marsac ◽  
Yaroslav de Kouchkovsky
Keyword(s):  
Carbon Source ◽  
Photosynthetic Pigments ◽  
Culture Conditions ◽  
Limiting Factors ◽  
Photosynthetic Oxygen Evolution ◽  
Autotrophic Growth ◽  
Heterotrophic Culture ◽  
Tobacco Cells ◽  
Liquid Suspension ◽  
Photosynthetic Oxygen

Two clones of Nicotiana tabacum in liquid suspension culture were grown photosynthetically using air plus 2% CO2 as carbon source, and agitation. In general, the transfer from heterotrophic to autotrophic growth in these cells was accompanied by an increase in the concentration of photosynthetic pigments, an increase in the N, C, and H contents and in the N:C ratio, an increase in the photosynthetic oxygen evolution capacity, and a decrease in the respiratory rate. The limiting factors are discussed.Several clones were also grown photosynthetically on agar slants and the development of their photosynthetic pigments studied. Results showed that one cannot accurately predict the photosynthetic capability of tobacco cells from their appearance in heterotrophic culture conditions.

scholarly journals Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

2011 ◽  
Vol 12 (11) ◽  
pp. 7732-7747 ◽  
Author(s):  
Simone Peletto ◽  
Simone Bertuzzi ◽  
Chiara Campanella ◽  
Paola Modesto ◽  
Maria Grazia Maniaci ◽  
...  
Keyword(s):  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Reference Genes ◽  
Internal Reference ◽  
Quantitative Expression

scholarly journals Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1253
Author(s):  
An-Pei Yang ◽  
Yu-Sheng Wang ◽  
Cong Huang ◽  
Zhi-Chuang Lv ◽  
Wan-Xue Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Tuta Absoluta ◽  
Pcr Analysis ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Real Time Quantitative Pcr ◽  
Adult Tissues ◽  
Induced Stresses

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

scholarly journals Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2763 ◽  
Author(s):  
Xiaofeng Wang ◽  
Jinting He ◽  
Wei Wang ◽  
Ming Ren ◽  
Sujie Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Laryngeal Cancer ◽  
Reference Genes ◽  
Target Gene ◽  
Cancer Tissue ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Internal Reference Gene ◽  
Relative Quantitation

BackgroundThe aim of this study was to determine the expression stabilities of 12 common internal reference genes for the relative quantitation analysis of target gene expression performed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human laryngeal cancer.MethodsHep-2 cells and 14 laryngeal cancer tissue samples were investigated. The expression characteristics of 12 internal reference gene candidates (18S rRNA, GAPDH, ACTB, HPRT1, RPL29, HMBS, PPIA, ALAS1, TBP, PUM1, GUSB, and B2M) were assessed by RT-qPCR. The data were analyzed by three commonly used software programs: geNorm, NormFinder, and BestKeeper.ResultsThe use of the combination of four internal reference genes was more appropriate than the use of a single internal reference gene. The optimal combination was PPIA + GUSB + RPL29 + HPRT1 for both the cell line and tissues; while the most appropriate combination was GUSB + RPL29 + HPRT1 + HMBS for the tissues.ConclusionsOur recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on laryngeal tumors.

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