Type-it Fast SNP Probe PCR Kit (4000)

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Publication protocol

Genotyping of IL1A –889 (rs1800587) and IL6 –174 (rs1800795, Intron type) SNP’s polymorphism was performed using a real-time PCR method. PCR amplification was done in a total volume of 25 µL, containing Type-it Fast SNP Probe PCR Master Mix (12.5 µL), primer–probe mix (1.25 µL), DNA (20 ng), and RNase-free water, by the following PCR cycling program: 1 cycle of 95 °C for 5 min for initial PCR activation, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s (Type-it Fast SNP Probe PCR Kit, Qiagen). After PCR amplification, end-point plate reading was completed on a real-time PCR instrument (Qiagen RotorGene Q, Qiagen, Hilden, Germany) based on fluorescence measurements. The allele detected by VIC was IL1A –889A (T) and IL6 –174C, the allele detected by FAM was IL1A –889G (C) and IL6 –174G. The primers GAT TTT TAC ATA TGA GCC TTC AAT G[A/G]T GTT GCC TGG TTA CTA TTA TTA AAG (IL1A –889), and ACT TTT CCC CCT AGT TGT GTC TTG C[C/G]A TGC TAA AGG ACG TCA CAT TGC ACA (IL6 –174) were used (TaqMan ®, ThermoFischer Scientific, Life Technologies Ltd, Paisley, UK).
APOE ε allele was determined via the hybridization method according to the manufacturer’s protocol (GenoType ApoE, ver. 1.0, 2015, Hain Lifescience GmbH, Qiagen, Nehren, Germany).

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