Total RNA was extracted from the formalin-fixed paraffin-embedded (FFPE) specimens of primary tumors by using Qiagen’s miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA).
Label
Nanostring
Label protocol
n/a
Hybridization protocol
n/a
Scan protocol
After hybridization, excess probes are washed away using a two step magnetic bead- based puri cation on the nCounter Prep Station. Magnetic beads derivatized with short nucleic acid sequences that are complementary to the Capture Probe and the Reporter Probes are used sequentially. First, the hybridization mixture containing target/probe complexes is allowed to bind to magnetic beads complementary to sequences on the Capture Probe. Wash steps are performed to remove excess Reporter Probes and non-target cellular transcripts. After washing, the Capture Probes and target/probe complexes are eluted o the beads and are hybridized to magnetic beads complementary to sequences on the Reporter Probe. An additional wash is performed to remove excess Capture Probes. Finally, the puried target/probe complexes are eluted o the beads and immobilized on the cartridge for data collection. After hybridization, excess probes are washed away using a two step magnetic bead- based puri cation on the nCounter Prep Station. Magnetic beads derivatized with short nucleic acid sequences that are complementary to the Capture Probe and the Reporter Probes are used sequentially. First, the hybridization mixture containing target/probe complexes is allowed to bind to magnetic beads complementary to sequences on the Capture Probe. Wash steps are performed to remove excess Reporter Probes and non-target cellular transcripts. After washing, the Capture Probes and target/probe complexes are eluted o the beads and are hybridized to magnetic beads complementary to sequences on the Reporter Probe. An additional wash is performed to remove excess Capture Probes. Finally, the purified target/probe complexes are eluted out the beads and immobilized on the cartridge for data collection.
Immune microenvironment of premenopausal estrogen receptor-positive/HER2-negative advanced breast cancer in response to treatment of Pembrolizumab, exemestane, and leuprolide
