Housekeeping gene selection in poplar plants under Cd-stress: comparative study for real-time PCR normalisation

2009 ◽  
Vol 36 (12) ◽  
pp. 1079 ◽  
Author(s):  
Brigitta Basa ◽  
Ádám Solti ◽  
Éva Sárvári ◽  
László Tamás
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Target Gene ◽  
Gene Selection ◽  
Developmental Stages ◽  
Plant Stress ◽  
Transcript Level ◽  
Cd Stress ◽  
Severe Stress ◽  
The Impact

Real-time RT–PCR is currently the most sensitive, specific and precise approach to analyse gene expression changes in plant stress studies. The determination of biologically meaningful transcript quantities requires accurate normalisation of the raw data. During relative quantification the reliability of the results depends on the stable expression of the endogenous control genes across the experimental samples. Four widely used internal control genes (cyclophilin, elongation factor 1α, polyubiquitin, tubulin β-chain) and two potential candidates (serine/threonine-protein phosphatase 2A and ubiquitin-conjugating enzyme) genes were assessed under Cd-stress and at different developmental stages in leaves of Populus jacquemontiana D. var. glauca H. Complementary DNA (RiboGreen) based quantification method revealed variations in the expression level of reference genes. The variability was more pronounced under severe stress conditions. Less variation was observed in the case of ef-1α, pp2a and ubc10. Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the RiboGreen method or by using multiple references. The impact of variability of reference gene on the target gene evaluation was demonstrated. It was proved that in the absence of suitable housekeeping genes, for example under severe stress, RiboGreen method is convenient tool for transcript normalisation.

scholarly journals An integrated genome-wide multi-omics analysis of gene expression dynamics in the preimplantation mouse embryo

2018 ◽  
Author(s):  
Steffen Israel ◽  
Mathias Ernst ◽  
Olympia E. Psathaki ◽  
Hannes C. A. Drexler ◽  
Ellen Casser ◽  
...  
Keyword(s):  
Mouse Embryo ◽  
Developmental Stages ◽  
Protein Complexes ◽  
Large Fraction ◽  
Transcript Level ◽  
Free Ribosome ◽  
Translational Machinery ◽  
Preimplantation Mouse ◽  
Morula Stage ◽  
The Impact

AbstractEarly mouse embryos have an atypical translational machinery comprised of cytoplasmic lattices, poorly competent for translation. Thus, the impact of transcriptomic changes on the operational levels of proteins has likely been overestimated in the past. To find out, we used liquid chromatography–tandem mass spectrometry to detect and quantify 6,550 proteins in the oocyte and in six developmental stages (from zygote to blastocyst) collected in triplicates, and we also performed mRNA sequencing.In contrast to the known split between the 2-cell and 4-cell stages at the transcript level, on the protein level the oocyte-to-embryo transition appeared to last until the morula stage. In general, protein abundance profiles were weakly correlated with those of their cognate mRNAs and we found little or no concordance between changes in protein and transcript expression relative to the oocyte at early stages. However, concordance increased towards morula and blastocyst, hinting at a more direct coupling of proteins with transcripts at these stages, in agreement with the increase in free ribosome abundance. Independent validation by immunofluorescence and qPCR confirmed the existence of genes featuring strongly positively and negatively correlated protein and transcript. Moreover, consistent coverage of most known protein complexes indicates that our dataset represents a large fraction of the expressed proteome. Finally, we identified 20 markers, including members of the endoplasmic reticulum pathway, for discriminating between early and late stages.This resource contributes towards closing the gap between the ‘predicted’ phenotype, based on mRNA, and the ‘actual’ phenotype, based on protein, of the mouse embryo.

Comparative Study Between Relative and Absolute BCR-ABL Transcript Quantification by Real Time PCR in Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4248-4248
Author(s):  
Paula Oliveira Montandon Hokama ◽  
Juliana Capannacci ◽  
Newton Key Hokama ◽  
Kozue Miyashiro ◽  
Fernando Lopes Alberto ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Internal Control ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Target Gene ◽  
Chronic Phase ◽  
Absolute Quantification ◽  
Standard Curve ◽  
The Absolute

Abstract After the introduction of tirosine-kinase inhibitor in chronic myeloid leukemia (CML) treatment, the quantification of the level BCR/ABL positive cells has become essential. Since 1993, our lab has been using a sensitivity qualitative nested RT-PCR assay. Due to the need of tumor cells quantification in CML, we developed, in 2004, a quantitative test by real time PCR. The question was to define whether the relative or absolute quantification was the best strategy. To answer this question our lab team standardized both methods and compared them. The real time PCR (RQ-PCR) was performed in Applied Biosystems® 7500 plataform with TaqMan® probes towards b2a2, b3a2 BCR-ABL transcript and BCR reference gene, in a non multiplex assay. Two separate RQ-PCR reactions were prepared for the BCR-ABL standard and BCR standard. 129 peripheral blood samples of CML patients were tested by both relative and absolute methods. Quality control samples were analyzed in every RQ-PCR run to monitor assay performance: NTC, positive and negative control. Relative Quantification is based on the expression levels of a target gene (BCR-ABL) versus a reference gene (BCR). This method determines the mRNA level of BCR-ABL gene across mutiple samples and expresses it relative to the levels of an internal control. A pool of c-DNA of 30 patients with untreated CML in chronic phase was used as an internal control (N Engl J Med, 2003, oct 9, 349–15). This method does not require standards with known concentrations and we used Pfaffl mathematical model to calculate the expression of a target gene in relation to a reference gene (Nucl Acid Res.2001; 29:2002–7). The relative expression ratio is calculated from the real time PCR efficiencies and the crossing point of an unknown sample versus a control: Ratio= E(target) ΔCt target (controlsample)/E(reference) ΔCt reference (control-sample) Absolute Quantification determines the input copy number of the BCR-ABL transcript, usually by relating the PCR signal to a standard curve. The standard curve was constructed using plasmids. Plasmids contaning a cDNA fragment of genes under analysis (b2a2, b3a2 BCR-ABL transcript and BCR gene) were prepared by PCR cloning (Branford S, Br J Haematol, 1999; 107:508–99). A 10-fold diluition series in the range of 106 to 10 copies was prepared for the BCR-ABL transcripts and BCR gene. The results were reported as a ratio of BCR-ABL/BCR % and were expressed relative to the median of BCR-ABL transcripts in the blood of 30 patients with untreated CML in chronic phase (baseline). Results: We performmed 129 blood samples of CML patients by both the relative and the absolute RQ-PCR. The results showed a positive correlation between relative and absolute values (r = 0.969 and p =0.000). Conclusion: The results of relative and absolute RQ-PCR methods were equivalent. Although the absolute method is more frequent in literature, the relative quantification presents more simply standardization, low contamination risk since it does not work with plasmids and results as safe as the absolute RQ-PCR.

Reference gene selection for quantitative real-time PCR normalization in different cherry genotypes, developmental stages and organs

2015 ◽  
Vol 181 ◽  
pp. 182-188 ◽  
Author(s):  
Xia Ye ◽  
Fangming Zhang ◽  
Yonghuan Tao ◽  
Shangwei Song ◽  
Jinbao Fang
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Gene Selection ◽  
Developmental Stages ◽  
Quantitative Real Time Pcr ◽  
Reference Gene Selection ◽  
Selection For

Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

2010 ◽  
Vol 37 (1) ◽  
pp. 12-19 ◽  
Author(s):  
Yael Brand ◽  
Ran Hovav
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Peanut Kernel ◽  
Tissue Samples ◽  
The Stability

Abstract Real-time qPCR is currently the most sensitive technique available for the detection of low-level mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1α, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginia-type peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra- and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.

scholarly journals Impact of Reference Gene Selection for Target Gene Normalization on Experimental Outcome Using Real-Time qRT-PCR in Adipocytes

PLoS ONE ◽  
2010 ◽  
Vol 5 (12) ◽  
pp. e15208 ◽  
Author(s):  
Bradley S. Ferguson ◽  
Heesun Nam ◽  
Robin G. Hopkins ◽  
Ron F. Morrison
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Target Gene ◽  
Gene Selection ◽  
Reference Gene Selection ◽  
Qrt Pcr ◽  
Gene Normalization ◽  
Selection For

The impact of real-time continuous glucose monitoring on metabolic control in children with type 1 diabetes

2020 ◽  
Author(s):  
Ruxandra Calapod Ioana ◽  
Irina Bojoga ◽  
Duta Simona Gabriela ◽  
Ana-Maria Stancu ◽  
Amalia Arhire ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Real Time ◽  
Metabolic Control ◽  
Continuous Glucose Monitoring ◽  
Glucose Monitoring ◽  
The Impact

THE IMPACT OF TV ADVERTISING ON VIRAL: ANALYSIS USING REAL-TIME AD RATINGS

2019 ◽  
Vol 2019 ◽  
pp. 790-791
Author(s):  
Cunhyeong Ci ◽  
◽  
Hyo-Gyoo Kim ◽  
Seungbae Park ◽  
Heebok Lee
Keyword(s):  
Real Time ◽  
Tv Advertising ◽  
The Impact

778-P: The Impact of Self-Management Blood Glucose (SMBG) Real-Time Decision Support on Glycemic Control in Adult with T1D in China

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 778-P
Author(s):  
ZIYU LIU ◽  
CHAOFAN WANG ◽  
XUEYING ZHENG ◽  
SIHUI LUO ◽  
DAIZHI YANG ◽  
...  
Keyword(s):  
Decision Support ◽  
Blood Glucose ◽  
Glycemic Control ◽  
Real Time ◽  
Self Management ◽  
The Impact

45. Education at distance: Broadcasting ECG rounds to Southeastern Ontario (BESO Project). An innovative approach for teaching electrocardiography

2007 ◽  
Vol 30 (4) ◽  
pp. 51 ◽  
Author(s):  
A. Baranchuk ◽  
G. Dagnone ◽  
P. Fowler ◽  
M. N. Harrison ◽  
L. Lisnevskaia ◽  
...  
Keyword(s):  
Health Care ◽  
Real Time ◽  
Digital Signal ◽  
Teaching Methodology ◽  
Care Providers ◽  
Long Distance ◽  
Medicine Clerkship ◽  
Time Problem ◽  
The Impact ◽  
Ecg Interpretation

Electrocardiography (ECG) interpretation is an essential skill for physicians as well as for many other health care professionals. Continuing education is necessary to maintain these skills. The process of teaching and learning ECG interpretation is complex and involves both deductive mechanisms and recognition of patterns for different clinical situations (“pattern recognition”). The successful methodologies of interactive sessions and real time problem based learning have never been evaluated with a long distance education model. To evaluate the efficacy of broadcasting ECG rounds to different hospitals in the Southeastern Ontario region; to perform qualitative research to determine the impact of this methodology in developing and maintaining skills in ECG interpretation. ECG rounds are held weekly at Kingston General Hospital and will be transmitted live to Napanee, Belleville, Oshawa, Peterborough and Brockville. The teaching methodology is based on real ECG cases. The audience is invited to analyze the ECG case and the coordinator will introduce comments to guide the case through the proper algorithm. Final interpretation will be achieved emphasizing the deductive process and the relevance of each case. An evaluation will be filled out by each participant at the end of each session. Videoconferencing works through a vast array of internet LANs, WANs, ISDN phone lines, routers, switches, firewalls and Codecs (Coder/Decoder) and bridges. A videoconference Codec takes the analog audio and video signal codes and compresses it into a digital signal and transmits that digital signal to another Codec where the signal is decompressed and retranslated back into analog video and audio. This compression and decompression allows large amounts of data to be transferred across a network at close to real time (384 kbps with 30 frames of video per second). Videoconferencing communication works on voice activation so whichever site is speaking has the floor and is seen by all the participating sites. A continuous presence mode allows each site to have the same visual and audio involvement as the host site. A bridged multipoint can connect between 8 and 12 sites simultaneously. This innovative methodology for teaching ECG will facilitate access to developing and maintaining skills in ECG interpretation for a large number of health care providers. Bertsch TF, Callas PW, Rubin A. Effectiveness of lectures attended via interactive video conferencing versus in-person in preparing third-year internal medicine clerkship students for clinical practice examinations. Teach Learn Med 2007; 19(1):4-8. Yellowlees PM, Hogarth M, Hilty DM. The importance of distributed broadband networks to academic biomedical research and education programs. Acad Psychaitry 2006;30:451-455

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