Transcriptor 1st strand cdna synthesis kit

Total RNA was extracted from total BM cells using ISOGEN (Nippon Gene, Tokyo, Japan) and 1 µg from each sample was reverse transcribed using a Transcriptor 1st Strand cDNA Synthesis kit (Roche Diagnostics, Tokyo, Japan). The ABL1 tyrosine kinase domain of BCR-ABL1 was amplified with nested RT-PCR with the following primers: 1st forward, (designed on BCR gene), 5′-TTCAGAAGCTTCTCCCTGCAT-3′ and reverse (located in ABL gene), 5′-CTTCGTCTGAGATACTGGATTCCT-3′; 2nd forward (located in ABL gene), 5′-AAGCGCAACAAGCCCACTGTCTAT-3′ and reverse (located in ABL gene), 5′-CTTCGTCTGAGATACTGGATTCCT-3′. Amplified fragments were directly sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Written informed consent was obtained from the patient in the present study, according to the Declaration of Helsinki.

Total RNA from BEAS-2B cells was extracted by TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (2 µg) was then reverse-transcribed into cDNA with the Transcriptor 1st strand cDNA Synthesis Kit (Roche Diagnostics). Real-time qPCR was performed using the OneStep TB Green RT-PCR Kit (Takara Bio, Inc.). The primers used are listed in Table I. The thermocycling program was 95˚C for 10 min followed by 40 cycles of 95˚C for 15 sec and 60˚C for 60 sec. The relative expression of each gene was calculated by the 2^-ΔΔCq method (13 (link)) and GAPDH was used as the internal control.

Total RNA was isolated by using high Pure RNA isolation kit (Roche), followed by the cDNA synthesis (using 700 ng total RNA) using Transcriptor 1st strand cDNA synthesis kit (Roche). The quantitative PCR (qPCR) assays were performed with SYBR green PCR master mix kit (Cat no. 4309155; Thermo Fisher Scientific GmbH, Schwerte, Germany) and qPCR machine (Quant Studio 6 Flex Real‐Time PCR System, Thermo Fisher Scientific). The expression of housekeeping gene GAPDH was used. Quantification of mRNA expression was performed using ΔΔC_T method. To check the effect of drug on sarcomere‐related gene expression, we employed the RT² Profiler PCR array system (cat no. PAMM‐099Z, Qiagen, Hilden, Germany) according to the manufacturer's instruction. The primer pairs were used for quantitative reverse transcription polymerase chain reaction (RT‐qPCR) to amplify cDNA, which are mentioned in the Supporting Information.