7500 fast dx real time pcr system

Total nucleic acid extraction was performed using the MagNA Pure Compact Nucleic Acid Isolation Kit 1 (Roche, Catalog# 03730964001) with a 200 μL elution volume, as per manufacturer’s guidelines for external lysis. All samples were heat inactivated in lysis buffer consisting of 280 μL MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche, catalog# 03246779001) with 20 μL Proteinase K (Roche, catalog# 03115828001) for 30 min at 56°C before extraction. Five μL of extracts were quantified by the PanR8 qPCR assay [9 (link)] with standard curve of positive control plasmid from 10,000 copies to 0.1 copies. All samples and standard curves were run in duplicate on a 7500 Fast Dx Real-Time PCR System (Applied Biosystems), and each plate included a standard curve, positive control, and no template controls.

Total RNA was extracted from cultured cells, exosomes derived from cell lines and PBMC of CML patients respectively using TRIzol reagent (Takara, Japan). Total RNA was reverse transcribed using Evo M-MLV RT Kit with gDNA Clean for q-PCR Ⅱ (Accurate Biology, China). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using TB Green™ Premix DimerEraser™ (Perfect Real Time) kit (Takara, Japan) on the Applied Biosystems™ 7500 Fast Dx Real-Time PCR system (Thermo, MA, United States). Relative expression levels of genes were normalized to GAPDH expression and calculated using the 2 −ΔΔC t method. All steps were conducted according to the manufacturer’s instructions. The sequence of circ_0058493: forward primer, TGG​GCT​TTC​CTG​TAC​CGA​AC; reverse primer, CTC​CGT​TGC​ATG​GCC​AGA​TA. The sequence of GAPDH: forward primer, CTG​AGA​ACG​GGA​AGC​TTG​TCA; reverse primer, CCA​GTG​GAC​TCC​ACG​ACG​TA.

The DNA was isolated from fresh peripheral blood using the PureLink Genomic DNA kit (ThermoScientific) according to the manufacturer instructions. The concentration and the purity (A₂₆₀/A₂₈₀) of the DNA was quantified by spectrophotometry (BioSpectometer basic, Eppendorf).
For genotyping of MC4R rs17782313 and ENPP1 rs1044498 we used TaqMan technology. In this respect we used the following single tub TaqMan SNP Genotyping assay formats: C__32667060_10 for rs17782313, and C__1207994_20 for rs1044498 (from ThermoFisher Scientific). Genotyping was performed according to the standard protocol for genotyping for 7500 Fast Dx Real-Time PCR System from Applied Biosystems. For genotypes interpretation the 7500 Fast Software v2.3 (Applied Biosystems) was used. All gDNA samples isolated from whole blood collected from children included in patient and control groups were successfully genotyped.

Total RNA was prepared using a Nucleospin RNA XS kit (Takara Bio) or RNeasy mini kit (Qiagen, Gaithersburg, MD, United States) and, subsequently, reverse transcribed to cDNA using the PrimeScript II Reverse transcriptase kit (Takara Bio). Viral mRNA levels were analyzed by qPCR using the 7500 Fast DX Real-Time PCR system (Applied Biosystems, Foster City, CA, United States) as previously described (Watanabe et al., 2015 (link); Sato et al., 2017 (link)). The original primer sequences used in this study are listed in Table 5.

Total RNA (10 ng) isolated from FFPE tissue samples were reverse transcribed using miRCURY LNA Universal RT cDNA synthesis kit II with RNA spike-in (Exiqon) in a 10 µL reaction volume for 60 min at 42 °C and 5 min at 95 °C, according to the manufacturer’s instructions. The reverse transcription product (cDNA) was diluted 100-fold and quantified by qRT-PCR using an ExiLENT SYBR Green master mix (Exiqon). qRT-PCR was performed in custom-made 96-well Pick & Mix microRNA PCR panel plates (Exiqon) and a 7500 Fast Dx Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). This PCR panel plates consisted of the 71 candidate genes (a single well for each gene) and the following quality controls: UniSp3 (triplicate), UniSp6 (single), and cel-miR-39 (single). The PCR protocol was applied as follows: incubation for 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 1 min at 60 °C, with a final melting curve analysis. The Ct values for qRT-PCR were determined using the SDS v1.4 software (Applied Biosystems) and the single-threshold method. Ct values that displayed unusual amplification curves (e.g., low amplification efficiency) were excluded from further analysis.

For DNA isolation, we used the manufacturer’s protocol of PureLink Genomic DNA kit (Thermo Fisher Scientific, Carlsbad, CA, USA) or Quick-DNA Miniprep Plus Kit (ZymoResearch, Irvine, CA, USA). Total DNA was purified from nucleated blood cells. Genotyping protocols used for TP53 rs1042522, MDM2 rs3730485, and MDM4 rs4245739 variants were previously published [21 (link),35 (link),36 (link),37 (link),38 (link)]. The protocols were in-silico checked using the following free tools: Primer-BLAST (National Center for Biotechnology Information, NCBI), in-silico PCR program (University of California, Santa Cruz, UCSC In-Silico PCR—UCSC Genome Browser), NEBcutter V2.0 for RFLP-PCR technique (New England BioLabs, Ipswich, MA, USA) and SNPCheck 3 program (National Genetics Reference Laboratories, NGRL Manchester). For MDM2 rs2279744 genotyping we used TaqMan assay (C__15968533_20, Thermo Fisher Scientific, Carlsbad, CA, USA) and 7500 Fast Dx Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The genotypes of 10% of the subjects were confirmed by capillary sequencing (3500 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA).
NCBI’s (NCBI Homo sapiens Annotation Release 109), Ensembl’s (Ensembl Release 98) and European Variation Archive’s genome browser were used for alleles annotation of the investigated variants.

We adopted TaqMan real-time PCR assays to genotype the selected five SNPs—rs4646903 (C_8879532_20), rs1048943 (C_25624888_50), rs1042522 (C_2403545_10), rs1801133 (C_1202883_20), and rs1801394 (C_3068176_10)—in cases and controls using a 7500 Fast Dx Real-Time PCR System (Applied Biosystems, Life Technologies Inc., USA). Eight negative controls and 104 DNA samples (cases and controls) were included in a 96-well plate to ensure the accuracy of genotyping results. We also repeatedly genotyped 10% of the samples, and the results were 100% concordant.