Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100331: HL60 and C2F8 cultured with WT1-c4 or PRAME-c3 (103 cells/100 µL/well + sgRNA (1 µM) x 16 well/DW, EGFP-m2 x 24 well/WT1-c4, PRAME-c3) ↓ cultured for 18h (顕微鏡下では変化なし) ↓ 100401: Total RNA extraction by using RNeasy Mini Kit (QIAGEN) ↓ 100401: Removal of genomic DNA by using TURBO DNase free kit (Ambion) ↓ (RNA 濃度を測定したが、検出できず) ↓ Synthesis of cDNA from total RNA by using Superscript® VILOTM cDNA synthesis kit (invitrogen) (通常、RNA 量を合わせて行なっているが、今回は RNA 溶出液を一律 7 µL 用いた) ↓ Quantification of WT1 and PRAME mRNA expression by real-time Q-PCR using KAPA SYBR FAST qPCR Kits (KAPA Biosystems) Primer pair for real-time Q-PCR:WT1 (133 bp) PRAME (116 bp) β-actin (111 bp) HL60 + EGFP-m2 sample, C2F8 + EGFP-m2 sampleを４倍段階希釈したものを用いて、同時に定量。 100331-0402 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100401: PCR 100402: PCR Retry ☆ High base line ☆ DW における蛍光の立ち上がりは無し。 ↓ primer, DW 等, 変えられるものは一新し て Retry 100401-02 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100405: HL60 and C2F8 cultured with WT1-c4 or PRAME-c3 (103 cells/100 µL/well + sgRNA (1 µM) x 16 well/DW, EGFP-m2 x 24 well/WT1-c4, PRAME-c3) ↓ cultured for 24h (顕微鏡下では変化なし) ↓ 100406: Total RNA extraction by using RNeasy Mini Kit (QIAGEN) ↓ 100406: Removal of genomic DNA by using TURBO DNase free kit (Ambion) ↓ (RNA 濃度を測定したが、検出できず) ↓ 100407: Synthesis of cDNA from total RNA by using Superscript® VILOTM cDNA synthesis kit (invitrogen) (通常、RNA 量を合わせて行なっているが、今回は RNA 溶出液を一律 5 µL 用いた) ↓ Quantification of WT1 and PRAME mRNA expression by real-time Q-PCR using KAPA SYBR FAST qPCR Kits (KAPA Biosystems) Primer pair for real-time Q-PCR:WT1 (133 bp) PRAME (116 bp) β-actin (111 bp) 100405-07 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100401: PCR 100402: PCR Retry ☆ High base line ☆ DW における蛍光の立ち上がりは無し。 ↓ cDNA 濃度が高すぎる可能性を考え、 cDNA を 10 倍希釈して Retry 100407 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100405 sample (cell + sgRNA cultured for 24h)を用いて再定量 100407: Synthesis of cDNA from total RNA by using TaKaRa RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa) (通常、RNA 量を合わせて行なっているが、今回は RNA 溶出液を一律 4.75 µL 用いた) ↓ Quantification of WT1 and PRAME mRNA expression by real-time Q-PCR using KAPA SYBR FAST qPCR Kits (KAPA Biosystems) Primer pair for real-time Q-PCR:WT1 (133 bp) PRAME (116 bp) β-actin (111 bp) 100405-07 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 Amplification Plot WT1 PRAME β-actin Melt Curve Gene Expression (Endogenous control: β-actin, Reference sample: + EGFP-m2) HL60 C2F8 1.2 1 1 0.8 0.8 RQ = 2-ΔΔCT RQ = 2-ΔΔCT 1.2 0.6 0.4 0.2 0.6 0.4 0.2 0 ■ WT1 ■ PRAME 0 DW EGFP m2 WT1 c4 PRAME c3 DW EGFP m2 WT1 c4 PRAME c3 100407 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 100331 sample (cell + sgRNA cultured for 18h) を用いて再定量 100408: Synthesis of cDNA from total RNA by using TaKaRa RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa) (通常、RNA 量を合わせて行なっているが、今回は RNA 溶出液を一律 4.75 µL 用いた) ↓ Quantification of WT1 and PRAME mRNA expression by real-time Q-PCR using KAPA SYBR FAST qPCR Kits (KAPA Biosystems) Primer pair for real-time Q-PCR:WT1 (133 bp) PRAME (116 bp) β-actin (111 bp) 100408 Effect of sgRNA on WT1 or PRAME mRNA expression in HL60 and C2F8 Amplification Plot WT1 PRAME β-actin Melt Curve Gene Expression (Endogenous control: β-actin, Reference sample: + EGFP-m2) HL60 C2F8 1.2 1 1 0.8 0.8 RQ = 2-ΔΔCT RQ = 2-ΔΔCT 1.2 0.6 0.4 0.2 0.6 0.4 0.2 0 ■ WT1 ■ PRAME 0 DW EGFP m2 WT1 c4 PRAME c3 DW EGFP m2 WT1 c4 PRAME c3 100408
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