For the identification of hotspot somatic mutations in TERT promoter, primer sets that amplify the hotspot sites of the TERT promoter were designed as previously described22 (link) and are available in our previously published study23 (link). PCR amplification was performed from 100 ng of genomic DNA using the AmpliTaq Gold 360 Master Mix Kit (Life Technologies) on a SimpliAmp Thermal Cycler (ThermoFisher) as previously described23 (link). Sequencing was performed using purified PCR fragments (QIAquick PCR Purification Kit, Qiagen) on an ABI 3730 capillary sequencer using the ABI BigDye Terminator chemistry (v3.1, Life Technologies). Sequences of the forward and reverse strands were analyzed using 4Peaks (https://nucleobytes.com/4peaks/ ). All analyses were performed in triplicate.
RNA extraction from FFPE tissues was performed using RecoverAll Total Nucleic Acid Kit for FFPE (ThermoFisher) according to manufacturer’s guidelines. Quantitative RT-PCR analysis was performed using SYBR Green. GAPDH was used as housekeeping genes for normalization. mRNA fold expression change was calculated by the 2-ΔΔCT method as previously described24 (link). The following Primers set were used: GAPDH Foward 5′ -AGGTGAAGGTCGGAGTCAACG-3′ and Reverse 5′ -TGGAAGATGGTGATGGGATTT-3′ and TERT25 (link) Foward 5′ -GCCGATTGTGAACATGGACTACG-3′ Reverse 5′ -GCTCGTAGT TGAGCACGCTGAA-3′.
RNA extraction from FFPE tissues was performed using RecoverAll Total Nucleic Acid Kit for FFPE (ThermoFisher) according to manufacturer’s guidelines. Quantitative RT-PCR analysis was performed using SYBR Green. GAPDH was used as housekeeping genes for normalization. mRNA fold expression change was calculated by the 2-ΔΔCT method as previously described24 (link). The following Primers set were used: GAPDH Foward 5′ -AGGTGAAGGTCGGAGTCAACG-3′ and Reverse 5′ -TGGAAGATGGTGATGGGATTT-3′ and TERT25 (link) Foward 5′ -GCCGATTGTGAACATGGACTACG-3′ Reverse 5′ -GCTCGTAGT TGAGCACGCTGAA-3′.
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