Frequently Asked Questions
Whole Exon Sequencing of RKO & HCT116 Wt Cell Lines
The downloadable Excel file and the two VCF files below contain the Variant information for the two wild type Colorectal Cell Lines used for the development of our CRISPR-engineered mutation cell lines.
Download Excel Download HCT116 VCF Download RKO VCF
Molecular, Cell, and Histological Reference Standards
Why use cell and molecular reference materials?
Reference materials for biologics provide a consistent and reliable resource for evaluating and optimizing various stages of your sample processing workflow. Whether you’re starting from DNA extraction, assay design, or library preparation, our reference materials can help you to identify and eliminate sources of variability within your protocols. Our reference standards for biologics is ideal for companion diagnostic assay development.
Molecular reference materials enables you to:
- Optimize your DNA extraction protocols prior to using valuable patient samples
- Monitor the impact of workflow changes on downstream results using consistent, third-party validated standards
- Establish limits of detection (LODs) using mixtures of isogenically-paired, mutant and wild-type DNA
- Utilize standardized materials to easily transfer workflows across labs or institutions
- Accurately evaluate batch-to-batch variability to streamline your quality control process
- Our reference standards for biologics are ideal for companion diagnostic assay development.
Applications:
Our cellular or molecular reference materials can be used for various histological or genetic analysis methodologies.
- Immunohistochemistry (IHC)
- H&E staining
- in situ hybridization (ISH & FISH)
- DNA sequencing (Sanger, Next-Generation Sequencing)
- PCR (quantitiative real-time or digital)
- DNA microarray
- RNA (gene-expression)
- QDP
Onco-Ref™ FFPE Cell Scrolls
What is the thickness of the FFPE Cell Scroll?
We prepare 20 µm sections using 5.0 mm diamater FFPE cell blocks.
How are you cell lines generated?
ASC uses CRISPR/Cas9 technology to enable precision genome editing in its MAPK mutation panel series. 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.
How do you validate mutational status in your cell lines?
All of our MAPK 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.
Do you offer mixtures of mutant and wild-type DNA?
We are currently working with collaborators to develop these products. Please inquire for additional information.
Is there a minimum number of scrolls that must be ordered?
Yes, 4 scrolls per mutation is the minimum order.
Onco-Ref™ Genomic DNA (gDNA)
How are your cell lines generated?
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.
How do you validate mutational status in your cell lines?
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 Onco-Reftm Isogenic Mutation Cell Line product datasheet. Click the specific mutation cell line and then download the product datasheet PDF.
How do you assess the quality of the gDNA?
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.
How do you quantify the gDNA(single mutation)?
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.
What is the contration of the gDNA?
The Onco-Reftm gDNA Reference Standards are normalized to 50ng/µl after quantification using the methods mentioned in FAQ question (above).
What does COSMIC stands for?
COSMIC is the acronym for Catalogue of Somatic Mutations in Cancer.
Do you offer mixtures of mutant and wild-type gDNA?
Yes. We proivde custom cell line or gDNA mixtures. We emply Digital PCR methods. Please inquire for more information.
What is the MAPK Pathway?
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.
What does homozygous mean?
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.
