NRAS StripAssay

The Viennalab NRAS XL StripAssay determines the NRAS mutation status of a tumour sample. The assay covers 22 mutations in codons 12, 13, 59, 60, 61 and 146, based on reference sequence NM_002524.4.

Clinical background

NRAS belongs to the RAS family of signal transduction proteins downstream of the EGFR receptor. A mutation keeps the pathway active regardless of the receptor, which is why patients with metastatic colorectal cancer carrying an NRAS or KRAS mutation do not respond to anti-EGFR antibodies. NRAS mutations also occur in melanoma, where they define a subgroup with its own treatment considerations.

Mutations covered

Codons 12 and 13 account for ten of the mutations, codons 59, 60 and 61 for eleven, and codon 146 for one: p.A146T (c.436G>A). The strip carries a control line for the PCR reaction, and PCR positive and negative controls are supplied for the three amplicons.

Method

The assay amplifies fragments of 101, 128, 134 and 204 bp with biotinylated primers and hybridises the products to allele-specific probes immobilised as parallel lines on a test strip. Bound product is detected with streptavidin-alkaline phosphatase and a colour substrate, and the pattern is read by eye against the supplied Collector sheet. Taq DNA Polymerase is supplied (75 U). DNA extraction reagents are not supplied; the DNA concentration has to be adjusted to 1 to 10 ng/µl. For DNA from formalin-fixed paraffin-embedded tissue, fluorometric quantification is preferable, because UV photometry overestimates the amount of amplifiable DNA.

• Amplification Mix (500 µl)
• Taq Dilution Buffer (500 µl)
• Taq DNA Polymerase (75 U)
• DNAT (1.5 ml)
• Typing Trays (3)
• Test Strips (20)
• Hybridization Buffer (25 ml)
• Wash Solution A (80 ml)
• Conjugate Solution (25 ml)
• Wash Solution B (80 ml)
• Color Developer (25 ml)
• Collector™ Sheet