Nucleotide sequence of the EGFRvIII mRNA transcript around the region of interest (120bp) was purchased as Ultramer RNA oligonucleotide (Integrated DNA Technologies). Amount of Oligo was 68.49 nmol and purified using standard desalting. The quality of the synthetic RNA was not optimal due to the high GC content. The ultramer RNA oligo was then resuspended in nuclease free water to make upto 100μM. Initial quantification using NanoDrop™ Spectrophotometer (ThermoFisherScientific) demonstrated RNA concentration of 4,430 ng/μl. Aliquots were prepared and stored at −80.0 °C. Accurate qualitative and quantitative analysis of the RNA was performed using Agilent RNA 6000 pico kit run on Agilent Technologies 2100 Bioanalyzer (Waldbronn, Germany). For this purpose, the sample was diluted 2000 fold (based on Nanodrop measurement). Electropherograms obtained from Bioanalyzer revealed two peaks and we manually selected for the concentration (area under the curve) of the peak of interest based on the RNA size (120bp). The resulting nanograms were then converted to EGFRvIII copy number using the standard copy number formula for single stranded RNA as shown below.
Where: X = amount of amplicon (ng)
N = length of ssRNA amplicon
660 g/mole = average mass of 1 bp ssRNA
Where: X = amount of amplicon (ng)
N = length of ssRNA amplicon
660 g/mole = average mass of 1 bp ssRNA