Array CGH analysis was performed using a SurePrint G3 Human CGH Microarray 8 X 60 K kit (Agilent Technologies, Santa Clara, CA, USA), which consisted of 62,976 oligonucleotide probes spaced at 41 kbp intervals (median probe spacing) throughout the genome. Control DNA (Promega Corp., Nepean, Canada) was used as the reference DNA. DNA digestion, labeling and hybridization were performed following the manufacturer’s instructions. Scanned images were quantified using Agilent Feature Extraction software (v10.0), and the resulting data were imported into Agilent Genomic Workbench 7.0.4.0 software for visualization, and copy number variations were detected using the Aberration Detection Method-2 (ADM-2) algorithm. All genomic coordinates were based on human genome build hg19/GRCh37.
Control dna
Manufactured by Promega
Sourced in United States
Control DNA is a laboratory reagent used to verify the performance of DNA-based analytical procedures. It provides a known reference sample to ensure the accuracy and reliability of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Salsa p107 mlpa kit, by MRC-Holland (2 mentions)
Genomic workbench 7, by Agilent Technologies (1 mentions)
Sureprint g3 human cgh microarray 8 x 60 k kit, by Agilent Technologies (1 mentions)
Feature extraction software v10.0, by Agilent Technologies (1 mentions)
Cyanine3 (cy3), by Agilent Technologies (1 mentions)
Kreapure columns, by Agilent Technologies (1 mentions)
5 protocols using control dna
1
Array CGH Analysis of Human Genome
2
Comparative Genomic Hybridization Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA samples from GC cell lines were labeled using the CGH Nick Translation Kit (Abbott Laboratories, IL, United States) with Control DNA (Promega, Madison, United States) according to the manufacturer’s instructions. Hybridization was performed with CGH Metaphase Target Slides (Abbott Laboratories, Illinois, United States), following the manufacturer’s protocols. The slides were analyzed by Corel Photo-Paint - Version 5.00 - Isis Zeiss® software, using an Axioskop Zeiss microscope (Carl Zeiss Inc. Canada, Don Mills, ON, Canada) equipped with an epi-illuminator and fluorochrome-specific optical filters.
The three-color images with red, green, and blue were acquired from 15 metaphases. Chromosome imbalances were detected on the basis of the deviation of the fluorescence ratio profile from the balanced value (FITC:rhodamine = 1). For each chromosome, the final ratio values were prepared from the mean values of at least ten chromosome homologues from separate metaphase spreads. The CGH results were plotted as a series of green to red ratio profiles.
The three-color images with red, green, and blue were acquired from 15 metaphases. Chromosome imbalances were detected on the basis of the deviation of the fluorescence ratio profile from the balanced value (FITC:rhodamine = 1). For each chromosome, the final ratio values were prepared from the mean values of at least ten chromosome homologues from separate metaphase spreads. The CGH results were plotted as a series of green to red ratio profiles.
3
Detecting Copy-Number Variations in D2HGDH
4
Detecting Copy-Number Variations in D2HGDH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect copy-number variations within the D2HGDH gene, we used MLPA and real-time semi-qPCR. Exons 2, 6, 7, 9 and 10 were analysed with the SALSA P107 MLPA kit (MRC-Holland), according to manufacturer's instructions. The copy number of exons 3, 4, 5, 8 and of a region 4 Mbp upstream to the gene's transcription start site was determined by relative quantification using qPCR. Tumour samples (n= 50, Supplementary Table 2 ) were compared with a commercially available control DNA (Promega, Madison) and normalized by a control PCR product on chromosome 22. Relative abundance (copy-number variation) was determined by the 2−ΔΔCT method31 (link).
5
Comparative Genomic Hybridization of FFPE DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Genome CGH Microarray 244A slides (Agilent Technologies, CA, USA) were used for FFPE extracted DNA samples. We followed the protocol described in [30 (link), 31 (link)]. A total of 2.5 μg sex matched control DNA (Promega, WI, USA) was fragmented by sonication. The FFPE DNA was fragmented only if there was any large molecular weight DNA. About 500 ng of the fragmented samples were then run on 1.5% agarose gel for 1 hour to check the extent of fragmentation of the DNA. Once fragmentation was deemed appropriate, 2 μg of the control DNA was labeled with Cy3 (Agilent technologies, CA, USA) and 2μg of FFPE DNA with Cy5 (Agilent technologies, CA, USA) for 30 minutes at 85°C. After labeling the DNA was purified using KREA pure columns (Agilent technologies, CA, USA). The samples were then measured on a Nanodrop and Degree of Labeling (DOL) was calculated according to the following formula;
The samples were hybridized onto microarray slides only if the DOL was between 1.5–2.5%.
The samples were hybridized onto microarray slides only if the DOL was between 1.5–2.5%.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!