Arcturusxt lcm

All diagnostic biopsies and RP specimens were retrieved for tumour sampling. Following pathological assessment, tumours were delineated and microdissection performed using the ArcturusXT LCM (Applied Biosystems, CA) system. Quality control of tissue sampling was performed using an in-house real-time polymerase chain reaction (RT-PCR) method, which assesses amplifiability on three housekeeping genes (actin beta, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase complex flavoprotein subunit A). RNA was amplified using RT-PCR via a targeted RNA approach (Ampliseq for Illumina Transcriptome Human Gene Expression Panel, CA) to study gene expression levels using an input RNA of ~10 ng. Sequencing was conducted using Illumina HiSeq 4000 sequencer (Illumina, CA). Data was processed and analysed using the Illumina Local Run Manager RNA module, from which counts per transcript were obtained and aggregated by summation per gene.

For cryosections lacking fluorophores, slides were stained and dehydrated as described previously^{41 (link),42 (link)}. Briefly, slides were fixed immediately in 75% ethanol for 30–60 sec, rehydrated quickly with water, stained with nuclear fast red (Vector Labs) containing 1 U/ml RNAsin-Plus (Promega) for 15 sec, and rinsed two more times with water before dehydrating with 70% ethanol for 30 sec, 95% ethanol for 30 sec, and 100% ethanol for 1 min and clearing with xylene for 2 min. tdT- and EGFP-labelled cryosections were not stained and instead began with the 70% ethanol dehydration step that also provided solvent fixation. After air drying, slides were microdissected immediately on an Arcturus XT LCM instrument (Applied Biosystems) using Capsure HS caps (Arcturus). The smallest spot size was used, and typical instrument settings of ~50 mW power and ~2 msec duration yielded ~25 µm spot diameters capturing 1–3 cells per laser shot.