title: BioMark-RTqPCR, 2
status: Public on Jan 29 2021
contact.city: Albuquerque
contact.first: Rajanikanth
contact.last: Vadigepalli
contact.email: rvadigepalli@salud.unm.edu
contact.state: PA
contact.address: 1 University of New Mexico
contact.country: USA
contact.institute: University of New Mexico
organism: Rattus norvegicus
description: PCR assay
manufacture_protocol: Pooled single cell samples were prepared for multiplex real-time quantitative polymerase chain reaction (RT-qPCR) with the Biomark HD system using VILO III (ThermoFisher) reverse transcription of RNA into cDNA followed by 22 cycles of pre-amplification (Taqman Pre-amp Master Mix) of the cDNA using the collection of primers that were utilized in the multiplex RT-qPCR. Each set of primers was designed to span introns when possible and was validated through in silico PCR (Primer BLAST, NCBI) and observation of a single band with the expected amplicon size on PCR gel using standard rat brain RNA. The quality of the results of the multiplex RT-qPCR was ensured through examination of the melt curves and standard curves.