Proving Single Copy Nucleic Acid Sensitivity
The HER-2 gene is amplified in the SKBR3 breast cancer cell line, but present in normal diploid copy number in HeLa cells. Analysis of HER-2 gene copy number in these model cell lines demonstrated the single copy sensitivity of this in situ hybridization method.
DNA in situ hybridization of HER-2 and IL-8 genes in HeLa (left) and SKBR3 (right) cells. As expected, two copies (dots) of the HER-2 gene were evident in each HeLa nucleus, whereas multiple copies of the HER-2 gene were apparent in SKBR3 nuclei. As a control, normal IL-8 gene copy number was seen in both HeLa and SKBR3 nuclei. For DNA detection, cells were first treated with RNAse, then DNA was denatured prior to in situ hybridization.
QuantiGene ViewRNA - How it Works
Step 1- Prepare Sample
Adherent cells on a solid surface are fixed and permeabilized.
Step 2- Hybridize Probe Sets
Gene-specific Probe Sets hybridize to target mRNAs. For clarity, only single oligonucleotide pairs are shown; however, a typical Probe Set contains twenty or more oligonucleotide pairs. Each Probe Set TYPE, for example TYPE 1, interacts specifically with a corresponding signal amplification system, for example PreAmp 1, Amp 1, Label Probe 1, to generate signal for visualization.
Step 3- Amplify Signal
Independent but compatible signal amplification systems enable simultaneous detection of multiple RNAs in a single assay. Distinct sets of Pre-Amplifier (PreAMP), Amplifier (Amp) and Label Probe (LP) molecules are used to detect different target mRNAs. A PreAmp molecule hybridizes to each Probe Set oligonucleotide pair, then multiple Amp molecules hybridize to each PreAmp. Finally, multiple LP oligonucleotides conjugated to fluorescent dyes hybridize to each Amp.
Step 4-Image
Target mRNAs are visualized using a standard fluorescence microscope or automated imaging platform.
Assay Specifications
