Kapa hifi hotstart readymix 2x

Total RNA from tumor material was extracted and reversed transcribed to cDNA, which was subsequently amplified by PCR as described by Picelli et al.^{19 (link)} The Poly(A) tail was hybridized with OligodT primer (5′-AAGCAGTGGTATCAACGCAGAGTACT₃₀VN-3′) by incubating 2 μL of total RNA with 1 μL of dNTP mix and 1 μL of oligodt30VN (100 μM) at 72°C for 3 minutes. Reverse transcriptase mix was prepared with 5 M betaine and locked nucleic acid–modified TSO (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′), and the reaction was carried out in a thermal cycler with a heated lid for 90 minutes at 42°C followed by 10 cycles at 50°C for 2 minutes and 42°C for 2 minutes. cDNA was further pre-amplified using 10 μM random IS PCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) with KAPA HiFi Hotstart Ready Mix (2X) (Kapa Biosystems, Wilmington, MA, USA) and thermocycled under following conditions: denaturation at 98°C for 3 minutes, followed by 18 cycles at 98°C for 20 seconds, 67°C for 15 seconds, extension at 72°C for 6 minutes, final extension at 72°C for 5 minutes. PCR products were purified using Agencourt® AMPure®XP kit (Beckman Coulter, Brea, CA, USA). cDNA concentration was determined using Qubit 3 fluorimeter utilizing Qubit dsDNA high-sensitivity assay kit (Life Technologies Corporation, Eugene, OR, USA). cDNA was diluted further to a concentration of 10 ng/μL before proceeding for dPCR assay.