The promoter and terminator were amplified with Phusion® High-Fidelity PCR Master Mix with HF Buffer using the primer pairs F SacI s promHXT7/R promHXbaIXbandand F terHXT7 BSalII/respectivelylI, respectively (Additional file 1: Table S4).
Briefly, 100 ng of total RNA was reverse transcribed using the RT primer mix provided by the kit, which contains one RT primer for each of the 35 different apoptosis-related genes and four control genes in a single multiplex reaction.
The 5' RACE cDNA and negative control were amplified with Advantage 2 PCR kit using the universal primer mix (UPM) from the SMART-RACE kit and ORAC 242 primer (Table 4).
gDNA template (previously prepared) was used for comparison using the same primer mix as the cDNA reaction.
HLA locus-specific amplification was performed in a thermal cycler using the amplification primer mix and template DNA.
A 1/4 dilution of the cDNA for each sample was used, along with the primer mix and 2X SYBR master mix (Finnzymes, Thermo Scientific, Loughborough, UK).
All PCR products were purified by using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) and sequenced using an ABI Prism sequence (Applera) with Big Dye reaction mixes, using the primers chosen for the initial amplification, and analyzed on the BioEdit biological sequence editor 5.0.6 (7).
To generate the P[w + SelG CG1840] transgene, a genomic fragment was amplified with Long PCR Enzyme Mix (Thermo Scientific) using the primers 5′-GT GGATCCAAAAATGGTCTTGTTCCACA-3′, 5′-CG GCGGCCGCAATTTGGCGGAAGATTCAAA-3′ with Oregon R genomic DNA as a template.
Real-time PCR of cDNA products was performed on a Stratagene Mx3005P thermal cycler with Quantifast SYBR Green PCR master mix (Qiagen) using the primers detailed in Table 1.
Relative amount of endogenous Mamld1 mRNA against B2m (β2-microglobulin) was determined by the TaqMan real-time PCR method using the probe-primer mix on ABI PRISM 7000 (Life Technologies) (Assay No.: Mm01293665_m1 for Mamld1; and Mm00437762_m1 for B2m).
