Abi universal master mix

RNA was extracted from AML cells and quantified using qPCR. The RNA extraction kit was supplied by Macherey-Nagel, Düren, Germany. Reverse transcription was done with MMLV-RT (Promega, Madison, WI, USA). Real-time PCR was performed on the ABI7500 Real-Time PCR Instrument using ABI universal master mix (Applied Biosystems, Austin, TX, USA) and gene specific probes Hs01104728_m1 (ABL1), Hs00180411_m1 (BMI1), Hs00923894_m1 (CDKN2A), Hs00159202_m1 (MN1), and Hs02758991_g1 (GAPDH).
Measurements for BMI1 and MN1 were normalized with ABL1 values, measurements for CDKN2A were normalized with GAPDH values (ddCt relative quantitation). Assays were performed in three or more independent experiments. Statistical analysis was done on GraphPad Prism software using two-tailed t-tests. Data are depicted in column bar graphs plotting mean with SD values.

Real time quantitative PCR was performed on the extracted DNA samples using a StepOne Plus Real Time PCR system (Applied Biosystems, Thermo Fisher Scientific) using a method adapted from (60 (link)). Amplification of HCMV DNA used glycoprotein B primers (61 (link)) and detection with a Taqman probe (60 (link)) (specific sequences are detailed in Table S2) mixed with ABI Universal Mastermix (Applied Biosystems, Thermo Fisher Scientific). The final assay volume comprised 25µl with a 5µl extracted DNA sample or control sample included. PCR cycling conditions were 2 minutes at 50°C, 10 minutes at 95°C and 45 cycles of 15 seconds at 95°C and 60 seconds at 60°C. All donor samples were screened in triplicate with a standard curve of 1 – 10⁴ HCMV genomes [WHO International Standard (62 )] and spiked positive and negative HCMV DNA controls from blood, saliva and urine were generated as previously described (50 (link)) and run alongside the unknown samples. HCMV DNA copies detected was calculated using the StepOne software (Applied Biosystems, Thermo Fisher Scientific) from the standard curve run cycle threshold (Ct) values and required a minimum of 2/3 positive wells to report a result expressed as HCMV copies per milliliter.