Hotstart taq

MGMT promoter methylation status was assessed as previously reported [21 (link)]. Bisulfite modification of DNA was performed using the EpiTect Kit (Qiagen). Two primers were used to amplify the MGMT promoter region: 5’- GTTTYGGATATGTTGGGATA-3’ and reverse: 5’-biotin-ACCCAAACACTCACCAAATC-3’. The PCR analysis was performed in duplicate in 40μl reaction volume containing 0.5 μl of 10 μM each primer, 4μl 10× buffer, 3.2μl of 2.5 μM dNTPs, 2.5 U hotstart Taq (Takara, Madison, WI), and 2 μl of 10μM bisulfite-treated DNA. The PCR conditions were as follows: 95°C–3 min; 40 cycles of 95°C–15 s, 52°C–30 s, 72°C–30 s; 72°C–5 min (ABI PCR system 9700). DNA was purified from the total PCR product using QIAamp DNA Mini Kit (Qiagen) and then subjected to pyrosequencing (PyroMark Q96 ID System (Qiagen)) using the primer 5’-GGATATGTTGGGATAGT-3’ in accordance to the manufacturer’s instructions. The methylation values obtained were averaged across the seven CpG loci tested within the MGMT promoter. The GBM samples were considered MGMT promoter methylated with an average methylation of >10%.

Detection of DI RNA of different segments (PB1, PB2, and PA) was done according to a previously described method (34 (link)). For comparisons, passaged CA4-DelNS1, commercial coadapted Flumist live attenuated influenza vaccine (2014–2015 season), plaque-purified cold-adapted H1N1 CA7 from Flumist, and passaged Flu B-DelNS1 were examined. RNA was extracted by using a viral RNA extraction kit (Qiagen). Viral RNAs were subjected to reverse transcription using a High-Capacity cDNA synthesis kit (Invitrogen) and Uni-12 (Flu A) or Uni-9 (Flu B). Segment-specific viral genes were amplified using Hot start Taq (TaKaRa) and long-segment-specific primers. The NS segment was also amplified as a control. PCR products were resolved by the use of 1.5% agarose gels and stained using gel red (Biotium). Images were captured by an Azure 150 system (Azure).

MGMT promoter methylation status was detected by DNA pyrosequencing as previously described [34 (link), 35 (link)]. Bisulfite DNA modification was performed using the EpiTect Kit (Qiagen). The following primers were used to amplify the MGMT promoter region: 5′-GTTTYGGATATGTTGGGATA-3′ (forward) and 5′-biotin-ACCCAAACACTCACCAAATC-3′ (reverse). The PCR analysis was performed in duplicate in a 40-μl reaction volume containing 0.5 μl each primer (using a 10-μM working solution), 4 μl 10 × buffer, 3.2 μl of 2.5 μM dNTP, 2.5 U hotstart Taq (Takara Bio, Madison, WI, USA), and 2 μl of 10 μM bisulphite-treated DNA. The reaction conditions were: 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 30 s; and 72 °C for 5 min (ABI 9700; Applied Biosystems, Foster City, CA, USA). DNA was purified from total PCR products using QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and subjected to pyrosequencing (PyroMark Q96 ID System; Qiagen) using the primer 5′-GGATATGTTGGGATAGT-3′ in accordance with the manufacturer’s instructions. The obtained methylation values were averaged across the seven tested CpG loci within the MGMT promoter. Samples were considered as having a methylated MGMT promoter if the average methylation was ≥10 %.