Determination of Copy Number of c-Myc Protein per Cell by Quantitative Western Blotting

1999 ◽  
Vol 269 (1) ◽  
pp. 66-71 ◽  
Author(s):  
Claudia Rudolph ◽  
Gerold Adam ◽  
Andreas Simm
Keyword(s):  
Western Blotting ◽  
Copy Number ◽  
Quantitative Western Blotting

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

1999 ◽  
Vol 40 (3) ◽  
pp. 101-112 ◽  
Author(s):  
Ping Fu ◽  
Paul Senior ◽  
Ross T Fernley ◽  
Geoffrey W Tregear ◽  
G.Peter Aldred
Keyword(s):  
Copy Number ◽  
Mammalian Cells ◽  
Rapid Determination ◽  
Competitive Pcr ◽  
Transgene Copy Number

scholarly journals Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

2012 ◽  
Vol 12 (3) ◽  
pp. 349-356 ◽  
Author(s):  
Daniel Lipiński ◽  
Joanna Zeyland ◽  
Andrzej Pławski ◽  
Ryszard Słomski
Keyword(s):  
Copy Number ◽  
Transgenic Animals ◽  
Promoter Sequence ◽  
Absolute Number ◽  
Transgenic Pigs ◽  
Cmv Promoter ◽  
Genetic Construct ◽  
Coding Sequence ◽  
The Absolute

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

scholarly journals Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

2018 ◽  
Vol 11 (1) ◽  
pp. 1-11
Author(s):  
Hossein Mahboudi ◽  
Negin Mohammadizadeh Heidari ◽  
Zahra Irani Rashidabadi ◽  
Ali Houshmand Anbarestani ◽  
Soroush Karimi ◽  
...  
Keyword(s):  
Internal Control ◽  
Copy Number ◽  
Quantitative Methods ◽  
Absolute Quantification ◽  
Relative Quantification ◽  
Absolute Quantitation ◽  
Standard Curve ◽  
Qpcr Quantification ◽  
Comparative Manner

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

scholarly journals Direct determination of retrotransposon transposition rates in Drosophila melanogaster

1994 ◽  
Vol 63 (2) ◽  
pp. 139-144 ◽  
Author(s):  
Sergey V. Nuzhdin ◽  
Trudy F. C. Mackay
Keyword(s):  
Drosophila Melanogaster ◽  
Transposable Element ◽  
Inbred Line ◽  
Copy Number ◽  
Direct Determination ◽  
Hybridization Analysis ◽  
Spontaneous Mutations ◽  
Insertion Sites

SummaryRates of transposition and excision of the Drosophila melanogaster retrotransposon elements mdg3, 297, Doc, roo and copia were estimated directly, by in situ hybridization analysis of their cytological insertion sites in 31 replicates of a highly inbred line that had accumulated spontaneous mutations for approximately 160generations. Estimated transposition rates of Doc, roo and copia were, respectively, 4·2 × 10−5, 3·1 × 10−3 and 1·3 − 10−3; no transpositions of 297 nor mdg3 were observed. Rates of transposition of copia varied significantly among sublines. Excisions were only observed for roo elements, at a rate of 9·0 × 10−6 per element per generation. Copy number averaged over these element families increased 5·9 %; therefore, in these lines the magnitude of the forces opposing transposable element multiplication were weaker than transposition rates.

scholarly journals Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Biochemistry ◽  
2018 ◽  
Vol 57 (40) ◽  
pp. 5769-5774 ◽  
Author(s):  
Narek Darabedian ◽  
John W. Thompson ◽  
Kelly N. Chuh ◽  
Linda C. Hsieh-Wilson ◽  
Matthew R. Pratt
Keyword(s):  
Western Blotting

Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number

1995 ◽  
Vol 96 (3) ◽  
Author(s):  
Maris Laan ◽  
Kristiina Gr�n-Virta ◽  
Armi Salo ◽  
Pertti Aula ◽  
Leena Peltonen ◽  
...  
Keyword(s):  
Copy Number ◽  
Solid Phase ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Fish Analysis
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