Onco-Ref™ Genomic DNA (gDNA)

The  Onco-Ref™ Genomic DNA (gDNA) Reference Standards represent biologically-relevant control material that can be used for diagnostic assay development or can be used in routine monitoring of assay performance in a CLIA laboratory environment.  The  Onco-Ref™ Genomic DNA (gDNA) Reference Standards can be directly incorporated into the sample processing workflows for assay optimization, evaluate assay sensitivity and specificity, and analyze the impact of workflow changes on downstream analysis. 

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MAPK Pathway - Reference Standards

Oncoref - reference standards

Derived from our genetically engineered isogenic cell lines, the ONCOREF™ Genomic DNA (gDNA) Reference Standards represent biologically-relevant controls for use in assay development and/or routine QC monitoring of assay performance. These standards can be implemented to optimize sample processing workflows (e.g. extraction efficiency, reagent lot changes, workflow step modifications) and to asses assay sensitivity (i.e. LOD) and specificity (i.e. cross reactivity).  

Key Features:

  • Most comprehensive MAPK mutation panel on the market
  • Well-characterized colorectal cancer cells lines: EGFR (RKO), KRAS (RKO), BRAF (HCT116)
  • Paired, isogenic wild-type cell lines to serve as an ideal control
  • All mutations are homozygous 
  • Normalized to 50 ng/µl concentration
  • Reference cell lines are expanded from single-cells, ensuring maximum homogeneity
  • Available in multiple formats, including FFPE cell slides, FFPE cell scrolls, and FFPE cell blocks
Need a Custom FFPE Block using your own cell line?  Visit our FFPE Custom Services page for more details.  
 
To learn more about our FFPE Reference Standards and other diagnostic products, WATCH our WEBINAR! 
Technical Details

High Quality Source of Bio-Relevant Material 

All of our ONCOREFtm engineered cell lines are verified using Sanger DNA sequencing.  The image below shows the Sanger sequencing results for an EGFR 18 base-pair deletion and wildtype isogenic cell line pair  (COSMIC ID: 12367, Cat# ASE-8007).

Genomic DNA extracted from the mutant cell line was tested with a qPCR assay that was developed in-house. 

ASE-8007 - Reference Standards

Image 1:  Shows the Sanger Sequence alignment between the CRISPR-Cas9 generated deletion for an EGFR Mutated Cell Line (ASE-8007).
The lower left section of image 1 shows a standard curve from 10-fold dilution (NC, 0.05ng, 0.5ng, 5. 50ng , 500ng) of the extraced gDNA (ASO-6007) tested in duplicates.  
The lower left panel graph shows an in-house developed Real-Time TaqMantm qPCR assay and linear regression showed perfect correlation (r2 = 1.00).
 
In addition, for every cell line we asses the quality of the genomic DNA using an agarose gel. See Image 2 below.  
   
ASO-6007-DNA-Degradation-Test
Image 2: Results of QC DNA integrety testing.  Lane 1 is a 1Kb ladder. Lane 2,3 and 4 shows results of  three replicates of 0.5µg gDNA input.  
Support Materials

ONCOREF™ Reference Standards: Application of CRISPR/Cas9 to the Generation of Isogenic Cell Lines and Reference Materials (October 2016)

2016-Oncoref-webinar

 

 

 

 

 

 

 

 

 

CRISPR/Cas9 is rapidly enabling the development of new tools for enhancing our understanding oncogenic mutations in cancer. In order to aid in advancing cancer diagnosis and treatment, Applied StemCell has recently engineered a series of 40 isogenic cell lines that feature diverse mutations in the MAPK pathway. These mutant lines are available as isogenic pairs for applications in lead compound discovery, or as FFPE and nucleic acid reference materials for assay development. This webinar will focus on ASC’s efforts in developing these research tools, as well as applications of the materials for the advancement of cancer research.

Highlights of this talk:

  • Overview of molecular reference materials 
  • Workflow and QC for ONCOREF™ cell line generation
  • Advantages of CRISPR-engineered molecular reference standards
  • Applications of reference materials in assay development
  • Q & A
FAQ

1. How are your cell lines generated?

Answer: We use CRISPR/Cas9 technology to enable precision genome editing.  This technology allows for footprint-free gene modification, meaning that you don’t have to worry about the presence of selection markers or other genomic footprints during the development of your assay technologies.

2. How do you validate mutational status in your cell lines? 

Answer: All of our mutation panel cell lines are expanded from single clones.  This ensures maximum homogeneity of the genetic profile.  After clonal expansion, we confirm the mutational status of the cell line using Sanger sequencing.  You can see the sequence alignment for both the mutant and the wildtype on the ONCOREFtm Isogenic Mutation Cell Line product datasheet. Click the specific mutation cell line and then download the product datasheet PDF.   

3. How do you assess the quality of the gDNA?

Answer: We use agarose gel electrophoresis to assess gDNA degradation and look for excess small fragments.  In addition we use a spectrophotometry (A260/A280)  to  assess for protein contamination and relase lots only if  within a range of 1.75 -1.85.

4. How do you quantify the gDNA (single mutation)?

Answer: We use multiple methods to determine qunatity.  A Nanodrop testing is initially perfromed to obtain a rough inidication of the quantity.  Then we follow up with the Progema QuantiFlour Double Stranded DNA assay and take an average of three reading.  Finally, we use a  real-time qPCR assay to amplify the GAPDH locus.

5. What is the concentration of the gDNA?

Answer: The ONCOREFtm gDNA Reference Standards are normalized to 50ng/µl after quantification using the methods mentioned in FAQ question #4 (above).  

6.  What does COSMIC stands for?

         Answer: COSMIC is an acronym for Catalogue of Somatic Mutations in Cancer.  

7. Do you offer mixtures of mutant and wild-type gDNA?

Answer:  Yes.  We proivde custom cell line or gDNA mixtures. We emply Digital PCR methods.  Please inquire for more information. 

8. What is the MAPK Pathway?  

Answer: The mitogen-activated protein kinase (MAPK) pathway plays a role in the regulation of gene expression, cellular growth, and survival. Abnormal MAPK signaling may lead to increased or uncontrolled cell proliferation and resistance to apoptosis.  The MAPK signaling pathway encompasses a series of signal transduction events that flow from the engagement of EGFR at the outer cell membrane, through KRAS, BRAF, MEK, and ERK.  MAPK signaling ultimately results in transcriptional activation of key genes that promote cellular proliferation, survival, differentiation, motility, and angiogenesis.  As such, the MAPK pathway is one of the most frequently activated pathways in cancer, and several drugs have pharmacogenomics profiles that depend upon the MAPK mutational status.

mapk-figure

9. What does homozygous mean?  

Answer:  Homozygous refers to a particular gene that has identical alleles on both homologous chromosomes. It is referred to by two capital letters (AA) for a dominant trait, and two lowercase letters (aa) for a recessive trait.

Homozygous allele

References
  1. Akinleye A, Furqan M, Mukhi N, Ravella P, Liu D (2013) MEK and the inhibitors: from bench to bedside. J. Hematol. Oncol. 6, 1–11
  2. Santarpia L, Lippman SM, El-Naggar AK (2012) Targeting the MAPK–RAS–RAF signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16, 103–119
  3. Relling MV, Evans WE (2015) Pharmacogenomics in the clinic, Nature 526, 343–350
Ordering
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