PCR and RT-PCR were performed using an AmpliTaq Gold 360 kit (Applied Biosystems, Foster City, CA, USA). Following purification with ExoSAP-IT (Affymetrix USB, Cleveland, OH, USA), Sanger sequencing was performed using a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) for screening TERT C228T and C250T mutations, and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) for screening other genes on an auto sequencer (3130xl Genetic Analyzer, Applied Biosystems, Foster City, CA) in the sequencing analysis. The primer sequences are shown (Additional file 1 Table S1).
Amplitaq gold 360 kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The AmpliTaq Gold 360 kit is a polymerase enzyme-based solution designed for use in polymerase chain reaction (PCR) amplification of DNA samples. It provides reliable and consistent DNA amplification performance.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark assay design software 2, by Qiagen (3 mentions)
Pyromark q24 pyrosequencer, by Qiagen (3 mentions)
Pyromark q24 1, by Qiagen (3 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Ez dnam kit, by Zymo Research (1 mentions)
Lmd7000, by Leica (1 mentions)
Pcr tubes, by Takara Bio (1 mentions)
Pyromark q24gold reagents, by Qiagen (1 mentions)
5 protocols using amplitaq gold 360 kit
1
Screening TERT and Other Gene Mutations
2
Validation of DNA Methylation Changes
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Pyrosequencing was used to validate DNAm at five selected genes that showed differential methylation (P<0.05, FDR corrected) between preterm infants at TEA and term controls: SLC7A5, SLC1A2, NPBWR1 and QPRT. APOL1 was included in validation studies because of its functional relevance and the significance value from the array was marginal (P=0.05). Bisulphite conversion was performed on 500 ng of genomic DNA with the EZ DNAm kit (Zymo Research, Freiburg, Germany). The converted DNA was amplified using the AmpliTaq Gold 360 kit (Applied Biosystems, Warrington, UK) with primers mapping to target regions containing CpGs assayed within the array. PCR primers were designed using PyroMark Assay Design Software 2.0 (Qiagen; https://www.qiagen.com ). Pyrosequencing was performed using PyroMark Q24Gold reagents on a PyroMark Q24 Pyrosequencer (Qiagen) according to the manufacturer's instructions. Data were extracted and analysed using PyroMark Q24 1.0.10 software (Qiagen). Background non-conversion levels were ~1–3%.
3
Laser Microdissection and Mutational Analysis
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Nineteen of the 87 cases (13 AITL, 1 nodal PTCL with TFH phenotype and PTCL-NOS/nodal PTCL with TFH phenotype) were analyzed by LMD, which was performed using LMD7000 (Leica). The cells being positive for either PD1 or CD20 were dissected and collected into 0.2-ml PCR tubes (Takara, Shiga, Japan) containing 20 μl of distilled water. Stained cells at approximately 100 000 μm2 were dissected and collected for each sample. Genomic DNA was extracted using the QIAamp DNA PFFE Tissue Kit (Qiagen) following the manufacturer's protocol. Then 1 μl of DNA was used for PCR under the following conditions: 95 °C for 15 min, 60 °C for 4 min, 72 °C for 4 min, 35 to 40 cycles at 95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and 72 °C for 10 min using the AmpliTaq Gold 360 Kit (Applied Biosystems, Foster City, CA, USA) with each primer set (Supplementary Table S6 ). PCR amplicons were used for amplicon-based sequencing and Sanger sequencing.
4
Pyrosequencing Validation of DNA Methylation
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Pyrosequencing was used to validate DNA methylation at five CpG sites within 200 bp of the TSS of the leucine-rich, glioma inactivated 1 (LGI1) gene and additionally at a number of CpG sites with a spectrum of high, low and intermediate methylation levels on the array (IL17C, SAGE1, MIR4493, MIR548M, CLDN9 and TACC3). Bisulphite conversion was performed on 1 μg of genomic DNA with the EZ DNA methylation kit (Zymo Research). The converted DNA was amplified using the AmpliTaq Gold 360 kit (Applied Biosystems, Warrington, UK) with primers mapping to target regions containing CpGs assayed within the array. PCR primers (Supplementary Table 1 ) were designed using PyroMark Assay Design Software 2.0 (Qiagen). Pyrosequencing was performed using PyroMark Q24Gold reagents on a PyroMark Q24 Pyrosequencer (Qiagen) according to the manufacturer's instructions. Data were extracted and analysed using PyroMark Q24 1.0.10 software (Qiagen). Background non-conversion levels were <3%.
5
Pyrosequencing of DNA Methylation in Buccal Swabs and Rat Cortices
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Pyrosequencing was used to quantify DNA methylation at individual cytosines (CpGs) in DNA from buccal swabs from babies and from DNA extracted from rat brains and cortical slices. Bisulphite conversion was performed on 500 ng of genomic DNA using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Converted DNA was amplified using the AmpliTaq Gold 360 Kit (Applied Biosystems, Thermo Fisher Scientific, Paisley, UK)
with primers mapping to target regions containing CpGs assayed within the array from our previous study (Sparrow et al., 2016) . For analysis of DNA methylation in rat cortices, bisulfite converted DNA was amplified by PCR with primers mapping to the homologous promoter regions of Lrg1 in human and rat (Table 1). PCR primers were designed using PyroMark Assay Design Software 2.0 (Qiagen, Manchester, UK) (Table 1). Pyrosequencing was performed using PyroMark Q24Gold reagents on a PyroMark Q24 Pyrosequencer (Qiagen, Manchester, UK) according to the manufacturer's instructions. Data were extracted and analysed using PyroMark Q24 1.0.10 software (Qiagen, Manchester, UK). Background non-conversion levels were ~1-3%. Shapiro-Wilk normality test and unpaired t-testing was performed using SPSS (IBM, Hampshire, UK).
with primers mapping to target regions containing CpGs assayed within the array from our previous study (Sparrow et al., 2016) . For analysis of DNA methylation in rat cortices, bisulfite converted DNA was amplified by PCR with primers mapping to the homologous promoter regions of Lrg1 in human and rat (Table 1). PCR primers were designed using PyroMark Assay Design Software 2.0 (Qiagen, Manchester, UK) (Table 1). Pyrosequencing was performed using PyroMark Q24Gold reagents on a PyroMark Q24 Pyrosequencer (Qiagen, Manchester, UK) according to the manufacturer's instructions. Data were extracted and analysed using PyroMark Q24 1.0.10 software (Qiagen, Manchester, UK). Background non-conversion levels were ~1-3%. Shapiro-Wilk normality test and unpaired t-testing was performed using SPSS (IBM, Hampshire, UK).
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