Computer assisted gel documentation system

Total RNA was isolated from placental tissues or BeWo cells using EZNA. Total RNA Kit (OMEGA, Georgia, USA) according to the manufacturer's instructions. Reverse transcription to synthesize cDNA was accomplished using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). PCR amplification of the cDNA was performed using specific mouse primers shown in electronic supplementary material, figure S1. PCR was performed in a BIO-RAD S1000 Thermal cycler (BIO-RAD). The cDNAs were amplified for 40 cycles. One round of amplification was performed at 95°C for 5 s, at 56°C for 30 s and at 72°C for 30 s (TaKaRa, Japan). The PCR products (20 µl) were resolved on 2% agarose gels (Biowest, Spain) in a 1× TAE buffer (0.04 M Trisacetate and 0.001 M EDTA) and with GeneGreen Nucleic Acid Dye (TIANGEN, China). The reaction products were visualized using a transilluminator (SYNGENE, UK) and a computer-assisted gel documentation system (SYNGENE). The sets of primers used for RT-PCR are provided in the electronic supplementary material, figure S1. The ratio between the intensity of the fluorescently stained bands corresponding to the genes and β-actin was calculated to quantify the level of the transcripts for those genes mRNAs [15 (link),22 (link)]. The RT-PCR result was representative of three independent experiments.

Total RNA was isolated from HH10 chick embryos using a Trizol kit (Invitrogen, USA) according to the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized to a final volume of 25 μl using SuperScript RIII first-strand (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed as described previously^{29 (link),30 (link)}. The sets of primers used for RT-PCR are described in the Supplementary Fig. 1. The PCR reactions were performed in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The final reaction volume was 50 μl, comprising 1 μl of first-strand cDNA, 25 μM forward primer, 25 μM reverse primer, 10 μl PrimeSTARTM Buffer (Mg2+ plus), 4 μl dNTP Mixture (TaKaRa, Japan), 0.5 μl PrimeSTARTM HS DNA Polymerase (2.5 U/μl TaKaRa, Japan) and RNase-free water. cDNA was amplified for 30 cycles. One round of amplification was performed at 94 °C for 30 s and then 30 s at 58 °C and 30 s at 72 °C. The PCR products (20 μl) were resolved using 1% agarose gels (Biowest, Spain) in 1× TAE buffer (0.04 M Trisacetate and 0.001 M EDTA) and 10,000× GeneGreen Nucleic Acid Dye (TIANGEN, China) solution. The resolved products were visualized using a transilluminator (SYNGENE, UK), and photographs captured using a computer-assisted gel documentation system (SYNGENE). Each of these experiments was replicated at least three times.

Total RNA was extracted from the cells and tissues using a Trizol kit (Invitrogen, USA). First-strand cDNA was synthesized to a final volume of 25 μL using a SuperScript RIII first-strand kit (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed using chick-specific primers. The primers sequences are provided in Supplementary Figure 1. The PCR reactions were performed using a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA) as previously described (Ahir and Pratten, 2014 (link)). The resolved PCR products were visualized using a transilluminator (SYNGENE, UK), and photographs were captured using a computer-assisted gel documentation system (SYNGENE). The intensity of the fluorescently stained bands was measured and normalized using Image Pro-Plus.