Quantstudio 7 flex detection system

A QuantStudio 7Flex Detection System (Applied Biosystems, Thermo Fischer Scientific, Inc., USA) was used to measure the mRNA expression of Bax, Caspase-3 and Bcl-2. The reactions for the synthesis of cDNA were carried out using an iScript One-Step RT-PCR Kit with SYBR Green (cat no: 170–8892; Bio-Rad, Inc., Hercules, CA, USA). Briefly, primers (Table 10) were added to the reaction mixture at a final concentration of 10 pM. Thus, 5 µL of each cDNA sample were added to a 20 µL PCR mixture consisting of 12.5 µL of 2 × iScript One-Step RT-PCR Kit with SYBR Green, 0.5 µL of primers for Bax, Caspase-3 and Bcl-2 as well as GAPDH (Macrogen, Seoul, Korea), and 7 µL RNase/DNase-free water (Qiagen, DE). The thermal cycling conditions for Bax, Caspase-3 and Bcl-2 were established as 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C and 30 s at 60 °C, and finally 10 s at 95 °C. The presence of a single melting temperature peak verified the specificity of each primer. The expression of a house-keeping gene, GAPDH, was used as an endogenous control for the current work.

Total RNA was isolated with RNeasy kit (Qiagen) following the manufacturer's instructions. Reverse transcription was performed using iScript™ cDNA Synthesis kit (Bio-Rad). Quantitative PCR was carried out on a QuantStudio 7 Flex detection system (Applied Biosystems) with Power SYBR green PCR master mix (Applied Biosystems). Each sample was analysed in triplicate. Primers used are available on request.

Total RNA was isolated with RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). Quantitative PCR was carried out on a QuantStudio 7 Flex detection system (Applied Biosystems) with the Power SYBR green PCR master mix (Applied Biosystems). Each sample was analysed in triplicate. Primer sequences are provided in Supplementary Table 1.

Total RNA was extracted from T-ALL cell lines and even venetoclax-resistant T-ALL cells, using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. The High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA) was used to synthesize cDNA according to the manufacturer’s instructions. RT-qPCR was run to assess gene expression using gene-specific qPCR primer assays (ThermoFisher Scientific, Waltham, MA, USA) and an Applied Biosystems QuantStudio 7 Flex detection system. Each sample was analyzed in quadruplicate, and gene expression was normalized to the endogenous controls such as GAPDH and β-Actin. Relative changes in gene expression were calculated with the help of the comparative Ct method. Different probes used for qPCR included BCL2, BCL2L1, BCL2L2, MCL1, GAPDH, and β-Actin. These probes were ordered from ThermoFisher Scientific, Waltham, MA, USA. Target gene expression levels were normalized against GAPDH and β—actin, and relative expression was determined using the ΔΔCt method.

All control and a subset of 230 PSP (PSP Series 1) participants had genotypes obtained in our prior study [8 (link)]. We genotyped all 973 LBD-NP and an additional 810 PSP (PSP Series 2) patients using the same methods.
DNA was extracted from blood using AutoGenFlexStar (AutoGen) and FlexiGene Chemistry (Qiagen) or brain using AutoGen 245T using standard protocols. Genotyping was performed using TaqMan assays (ABI3_rs616338, C_2270073_20; PLCG2_rs72824905, C_97909430_10) following manufacturers’ protocol, using a QuantStudio 7 Flex Detection System with a 384-Well Block Module (Applied Biosystems, Foster City, CA).
All minor allele carriers (ABI3_rs616338-T, PLCG2_rs72824905-G) were confirmed using Sanger Sequencing. Polymerase chain reaction (PCR) primers with the following sequences were used to amplify and sequence the genomic region flanking the mutations: ABI3 5′-CTTCCTGCTCGCACCCGAC-3′, 5′-CTAATGCAGCATCCCCAACT-207 3′, PLCG2 5′- CCATAAATGAGGGCTCTCAG-3′, 5′-CATACCCACCTCACCCTTGT-3′. PCR products were purified using the Agencourt AMPure protocol (Beckman Coulter, CA) and sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were purified using Agencourt CleanSEQ (Beckman Coulter, CA) and run on an ABI33730xl Genetic Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher 4.8 (Gene Codes Corporation, MI).

Total RNA in the lung was extracted by the Fast Pure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China), from which cDNA was synthesized with the HiScript® II Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China) and quantified using the ChamQ universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). The results were analyzed using the QuantStudio 7 Flex detection system (Applied Biosystems Co., United States) and the 2^−ΔΔCT method to assess the levels of mRNAs encoding targeted genes normalized to GAPDH. The primers are shown in Table 3.