Animal tissue total rna extraction kit

We used the Animal Tissue Total RNA extraction kit from TianGen Biotech Co., Ltd. to extract total RNA from Wulong geese ovaries. The RNA was reverse transcribed using the Prime Script™ RT reagent Kit from TaKaRa Biosciences. The reverse transcription system is described in Supplementary Table 2. The primer sequences (Supplementary Table 3) were designed using Primer 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. qRT-PCR was performed on a CFX96 real-time system (Bio-Rad, Hercules, CA, USA); the reaction system and conditions are shown in Supplementary Tables 4 and 5. Three biological replicates and three technical replicates were used for each sample. The relative expression levels of each gene were calculated using the 2^−ΔΔCt method, with GAPDH as the reference gene. Gene expression bar charts were plotted using GraphPad Prism 7. Independent sample t-tests were performed using SPSS 26.0 statistical software.

Total RNA was extracted from intestine tissues using an Animal Tissue Total RNA Extraction Kit (TIANGEN, Beijing, China). Complementary DNA was synthesized from total RNA using poly (A)-tailed total RNA and reverse transcription primers with ImPro-II Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocol. qRT‒PCR was performed according to the instructions of Fast Start Universal SYBR Green Master Mix (Rox) (Roche Diagnostics GmbH, Mannheim, Germany). Relative transcriptional folds were calculated as 2^−ΔΔCt. GAPDH was used as an internal control for normalization. The primers are listed in Supplementary Table 2.

We extracted total RNA of tissues from spleen, heart, kidney, lung, liver and muscle of Fat-1 and IGF-1 knock-in piglets by Animal Tissue Total RNA Extraction Kit (TIANGEN, Beijing, China). 1ug of total RNA from each tissue was converted into cDNA by using the FastKing one-step RT-PCR kit (TIANGEN, Beijing, China). The cDNA from each tissue was used as template of real-time fluorescence quantitative PCR to quantify the expression of the IGF-1 and Fat-1 genes at the transcription level. Porcine GAPDH was selected as reference gene for real-time PCR, and the primers for IGF-1, Fat-1 and GAPDH were as following: Fat-1 forward strand, 5′-ACCACATCGACAAGGACCAC-3′ and reverse strand 5′-TGATGACGCACTGCACTCTT-3′; IGF-1 forward strand 5′-TGCTTGCTCTCCTTCACCAG-3′ and reverse strand 5′-TCCAGCCTCCTCAGATCACA-3′; and GAPDH forward strand 5′-GCCATCACCATCTTCCAGG-3′ and reverse strand 5′-TCACGCCCATCACAAACAT-3′. The real-time PCR was performed on Bio-Rad IQ5 instrument.