Sds version 2

To determine relative copy numbers of de novo insertions L1-dn13 and L1-dn14 within the hiPS-SB4 culture, we applied real-time qPCR using TaqMan fluorogenic probes. To this end, genomic DNA was isolated using 1 ml DNAzol Genomic DNA Isolation Reagent (MRC Inc, Cincinnati, OH, USA) from 1 × 10⁶ cells, according to the manufacturer’s instructions. A total of 100 ng of genomic DNA was used for quantitative real-time PCR (qPCR). Primer and probe combinations specific to the genomic 5′ junctions of the de novo insertions L1-dn13 and L1-dn14 (Fig. 3b) were used to quantify the copy number of the respective insertion in hiPSC cultivars. Each probe was labelled with flourochrom6-carboxyfluorescein and a non-fluorescent quencher. For normalization the single-copy gene RPP25 (Ribonuclease P/MRP 25kDa subunit; FAM/non-fluorescent quencher, primer limited, HS00706565_S1; Applied Biosystems) was used. Cycling conditions were: 95 °C for 15 min (one cycle), 95 °C for 15 s and 60 °C for 1 min (40 cycles). For analysis of real-time and end point fluorescence, the software SDS version 2.3 as well as RQ manager 1.2 (Applied Biosystems) were used.

Total RNA was isolated from U2OS cells using Trisure reagent following the manufacturer’s protocol (Bioline). Total RNA (2 μg) was reverse-transcribed using the high capacity cDNA Reverse Transcription kit (Applied Biosystems). PCR amplification reactions were performed with the ABI Prism 7900 HT Fast Real-Time PCR System. Applied Biosystems’ TaqMan Gene Expression Assays were used to quantify gene expression of MKK3 (Hs00177127_m1) and the housekeeping gene TBP (HS99999910_m1), which was used to normalize. Data from PCR were acquired and analyzed using Sequence Detector software (Applied Biosystems, SDS version 2.3).

The allele discrimination of SNP variants was performed using commercial TaqMan probes (Applied Biosystems, CA, USA) at a 20X concentration. We selected three SNPs: rs13118928 (commercial probe id: C__11375931_20), rs1828591 (C__11482211_10), and rs13147758 (C___2965080_10). These SNPs are located in intronic (noncoding) regions. Supplementary Table S4 summarizes the principal characteristics of the assessed SNPs.
Genotyping was evaluated by applying real-time PCR (qPCR) in a StepOne Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific Inc., Singapore), and genotype assignment was performed by sequence detection software (SDS) version 2.3 (Applied Biosystems, CA, USA).

Viremia in experimentally infected animals was assessed by qRT-PCR as already described (57 (link), 69 (link)). Briefly, blood samples (500 μl) were pretreated with 1 ml cold distilled water on ice for 10 min and then centrifuged at 4°C for 10 min at 13,000 × g. Armored RNA (Asuragen, USA) was added to each sample before RNA extraction and used as an internal control to verify RNA extraction efficiency. Total RNA was extracted from the resulting cellular pellet, using the High Pure nucleic acid extraction kit (Roche, Nutley, NJ), in accordance with the manufacturer's instructions. The quality of the samples was further assessed by amplifying the sheep β-actin gene as previously described (71 (link)). For each sample, 250 ng of RNA was used in a one-step qRT-PCR employing primers/probes for segment 5 (encoding NS1) of BTV and the armored control RNA. Samples were analyzed using a 7900HT fast real-time PCR system and the sequence detection system software SDS, version 2.3 (Applied Biosystems). BTV genome copy numbers expressed as log₁₀/μg of total RNA were derived using a standard curve generated from the amplification of in vitro transcribed synthetic BTV segment 5 RNA using the mMESSAGE mMACHINE T7 Ultra kit (Ambion), according to the manufacturer's instructions. Signal levels with threshold cycle (C_T) values of ≥40 were considered negative.

Fifty-five paired OSCC tumor and pericancerous normal tissues were homogenized in liquid nitrogen using a mortar and pestle and incubated with RNAzol B reagent (Tel-Test, Friendwood, TX), and total RNA was extracted according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg of total RNA and mixed with a reaction mixture consisting of commercially available primers (EGFR: Hs01076090_m1, INHBA, Hs00171410_m1, CDH1: Hs01023895_m1, CDH2: Hs00983056_m1, SP1: Hs00916521_m1, GAPDH, Hs99999905_m1; Assay-on-Demand, Applied Biosystems, Foster City, CA), RNase-free water, and TaqMan Universal PCR Master Mix. Quantitative real-time RT-PCR was performed and analyzed using a 7900 HT Sequence Detection System and SDS version 2 (Applied Biosystems, Foster City, CA), respectively. All experiments were performed in triplicate, and the mean fold change was calculated for each sample.