Stepone rt pcr machine

RNA was extracted using Trifast reagent (Euroclone), according to the manufacturer’s instructions. 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). cDNA was used to determine transcript levels by qRT-PCR in a StepOne RT-PCR machine (Applied Biosystems, Foster city, CA, USA) using SYBR® Select Master Mix (Life Technologies). Primers used are listed in Supplementary Table S3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as endogenous control was amplified with primers 5’-GAG AAG GCT GGG GCT CAT TT (forward) and 5’-AGT GAT GGC ATG GAC TGT GG (reverse). Data were analyzed using the ΔΔCT method. ΔC_t was calculated subtracting the average C_t value of GAPDH as control to the average C_t value of each transcript for PC3 and LnCaP. ΔΔC_t is the difference between the ΔC_t for each transcript for PC3 and the ΔC_t of each transcript for LnCaP as control. The reported fold expression, expressed as RQ (relative quantity), was calculated by 2^−ΔΔCt. The analysis was repeated three times in triplicate. The mean ± standard deviation (SD) of a representative experiment is reported (* p < 0.05).

RNA was extracted using Trifast reagent (Euroclone), according to the manufacturer’s instructions. 1 μg of RNA was reverse-transcribed into cDNA using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). cDNA was used to determine transcript levels by qRT-PCR in a StepOne RT-PCR machine (Applied Biosystems, USA) using SYBR^® Select Master Mix (Life Technologies). Primers used are listed in Supplementary Table 1. GADPH used as endogenous control was amplified with primers 5′-GAG AAG GCT GGG GCT CAT TT (forward) and 5′-AGT GAT GGC ATG GAC TGT GG (reverse). Data were analysed using the ΔΔCt method. ΔCt was calculated subtracting the average Ct value of GADPH as control to the average Ct value of each transcripts for Mel501 and Mel 501ac. ΔΔCt is the difference between the ΔCt for each transcript for Mel501ac and the ΔCt of each transcript for Mel501 as control. The reported fold expression, expressed as RQ (Relative Quantity), was calculated by 2-ΔΔCt. The analysis was repeated three times in triplicate. The mean ± SD of a representative experiment is reported (*p < 0.05).

cDNA was prepared from RNA that was used in the sequencing analysis using SuperScript II Reverse Transcriptase (Invitrogen). qPCR was performed on an Applied Biosystems StepOne RT-PCR machine using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and a TaqMan probe and primer set designed against chicken Sim1 (Thermo Fisher Scientific). 5 ng cDNA was used per reaction (20 μl volume) with cycle conditions of 95°C for 20 s, followed by 32 cycles of 95°C for 1 s and 60°C for 20 s. All reactions were carried out in triplicate and normalized against eukaryotic 18S rRNA expression (Thermo Fisher Scientific). Standard errors of the means were generated from the triplicate C_T values. Unpaired t-tests measured significance of expression change between appropriate samples. Applied Biosystems StepOne Software V2.3 was used to analyse the data and generate gene expression comparisons.

Ten whole limb buds at 0, 6, 12 and 24 hours were dissected from either quail or chick embryos. Total RNA was extracted using TRIzol™ Reagent (Life Technologies), purified using a Direct-zol RNA kit (Zymo Research) and cDNA prepared using SuperScript III Reverse Transcriptase (Invitrogen). qPCR was performed on an Applied Biosystems StepOne RT-PCR machine using SYBR Green Master Mix (Thermo Fisher Scientific) and a primer set for Cyp26b1 was designed against a sequence which was present in both chicken and quails, spanning exon junctions (Thermo Fisher Scientific). 5 ng cDNA was used per reaction (20μl volume) with cycle conditions of 95 °C for 20 sec, followed by 32 cycles of 95 °C for 1 sec and 60 °C for 20 sec. All reactions were carried out in triplicate and average C_T values normalized against eukaryotic 18S rRNA endogenous control expression (Thermo Fisher Scientific).