Rna qubit

Biopsy specimens were sent as rolls from FFPE when the estimated tumor percentages were greater than 50% or as slides, which were macrodissected when the tumor percentage was less than 50%. Total nucleic acids were extracted from the tissue using Agencourt FormaPure (Beckman Coulter). Complementary DNA was synthesized from material equivalent of 200 ng of RNA based on the RNA Qubit (Thermo Fisher Scientific) reading. PCR primers were designed to capture wild-type EGFR spanning exons 1 and 2, EGFRvIII spanning exons 1 to 8, and three housekeeping genes (HPRT, SDHA, and RPL13A). The assay also includes three primer sets with increasing target sizes built in the assay; this allows for assessment of the RNA degradation level of the sample in a single assay [exons 9 to 9, 93 base pairs (bp); exons 9 to 10, 141 bp; exons 9 to 12, 251 bp]. The NGS library preparation is a two-step PCR method: The first step is a multiplex PCR followed by second PCR to add Illumina sequencing index and adaptors. Subsequently, the sequencing library is quantitated on TapeStation (Agilent) and then sequenced on MiSeq (Illumina). A custom bioinformatics pipeline was developed to process the data. EGFRvIII ratio is calculated by the following formula built in the bioinformatics pipeline: EGFRvIII ratio = (EGFRvIII reads)/(EGFRvIII reads + WT EGFR reads).