Iq5 icycler realtime pcr detection system

RNA extraction was performed using an Aurum Total RNA kit (BioRad, USA) according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using oligo dT and random hexamers with Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCR amplifications were performed in an iQ5 iCycler real time PCR detection system (Bio-Rad) using the iQ SYBR Green supermix (Bio-Rad). qRT-PCR was performed with primer sets given in S1 Table, using the following cycles: an initial cycle at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s each, 59°C for 30 s, and 72°C for 30 s, followed by melt- curve analysis from 61–95°C in 0.5°C increments. mRNA expression was normalized to expression of B-2-microgoblulin (B2m) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the relative expression of target genes was obtained by the 2^-ddCt method [85 (link)]. A range of 3–14 biological replicates (individual hearts between the ages of E10-13.5, brain or kidney at E17.5) were assessed in technical duplicates or triplicates for each gene. Statistical significance was determined using Student’s t test (with Bonferroni correction where indicated).

Total RNA from cell lines and tumor samples was extracted with Trizol (Invitrogen) and RNaesy Extraction Kit (QIAGen) as indicated by the manufacturer. cDNA from the different cell lines and tumor samples was obtained from 1 μg of total RNA using random primers and Superscript II system (Life Technologies Inc) as previously described [13] . Gene expression analyses were performed by semiquantitative RT-PCR (sqRT-PCR) and real time RT-PCR (qRT-PCR). For qRT-PCR pre-designed TaqMan probes (GSDMB, GSDMB-1, GSDMB-2, GSDMB-3&4) or SybrGreen PCR reagents (mCherry) (Sigma) were used on an iQ5 iCycler Realtime PCR Detection System (BioRad) using TaqMan “iQ Supermix” or “SYBR Green Supermix” (BioRad), according to the manufacturer's recommendations. GSDMB-3 and -4 were analyzed together as there is no commercially available Taqman probe to discriminate isoform 3. Primers sequences and amplification conditions for sqRT-PCR and for qRT-PCR are indicated in Tables S1 and S2, respectively. All RT-PCRs were performed in triplicates. Relative expression was normalized to β2 microglobulin, β-actin or GAPDH. The comparative threshold cycle (Ct) method was used to calculate the amplification factor as specified by the manufacturer.

Gene expression was performed after extraction of total RNA from PDXs using RNA miniprep system, ReliaPrep^TM FFPE total (Promega) following manufacturer recommendations. cDNA was obtained from 1 μg of total RNA using random primers and M-MLV reverse transcriptase (Amresco) as manufacturer recommendations. GSDMB, HER2, and GAPDH gene expression levels were measured by quantitative real time RT-PCR (qRT-PCR) using pre-designed TaqMan probes (Thermofisher) and PerfeCTa FastmixII (Quanta BioSciences, Inc), on an iQ5 iCycler Realtime PCR Detection System (BioRad), according to the manufacturer's recommendations. All qRT-PCRs were performed in triplicate. Relative GSDMB and HER2 expression was normalized to GAPDH.