Realmastermix sybr green 1

To verify the accuracy and repeatability of the RNA-seq results of DEGs, 10 genes were selected to measure expression levels in different follicles using qRT-PCR. The primers used for amplification of the candidate genes, including anti-Müllerian hormone (AMH), cyclin O (CCNO), cadherin 3 (CDH3), follistatin (FST), and NPC intracellular cholesterol transporter 2 (NPC2), were designed using the Primer-Blast tool in the National Center for Biotechnology Information (NCBI) database and are listed in Supplemental Table S1. The cDNA was synthesized in 2 µg total RNA using a FastKing gDNA Dispelling RT SuperMix (Tiangen, Beijing, China). qRT-PCR was performed on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) using Real Master Mix SYBR Green I (Tiangen, Beijing, China). The total reaction volume was 20 µL, which comprised 1 µL cDNA, 10 µL 2 × Talent qPCR PreMix, 0.6 µL each of the forward and reverse primers, and 7.8 µL RNase-free water. The thermocycling program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. The $2^{-△△ct}$ method was used to calculate gene expression levels [18 (link)]. Expression levels of coding genes were normalized to those of β-actin.

Four genes (MFSD12, TYR, NR1H4 and THRSP) were selected to measure expression levels in feather follicles of 7- and 11-week old chickens, in order to verify the RNA-seq data using qRT-PCR. Five key pigment genes (TYRP1, DCT, PMEL, MLANA and HPGDS) were specifically chosen to assess their function in melanogenesis; to this end, we measured their expression levels in all samples of 3-, 5-, 7-, and 11-week old chickens. The primers for these genes are described in Table S5.
The qRT-PCR was performed on a CFX96TM Real-Time System (Bio Rad, Hercules, CA, USA) using Real Master Mix SYBR Green I (Tiangen). GAPDH was used as a housekeeping gene in all qRT-PCR experiments. Five biological replicates per group and three technical replicates per sample were used for qRT-PCR. The 2^−ΔΔCT method was used to calculate gene expression levels^{74 (link)}. Significant differences between the two groups were evaluated using unpaired Student’s t tests. All results are shown as the means ± standard error (SE). P < 0.05 was considered significant and P < 0.01 extremely significant.

Eight male ducks from each group were selected randomly for qRT-PCR analysis. Eight genes, including four (MYOG, CAV3, IGFBP5, and TNNI1) that showed similar differential expression patterns, and four (ACSL1, MSTN, CD36, and FHC3) that showed different expression patterns between the breeds, were selected for the validation of the differences in expression as revealed by RNA-seq by using qRT-PCR. The primers were designed considering the basic principles for qRT-PCR primer designing (see Supplementary Table S8). The standard curve was prepared using calibrated samples with five-fold dilutions. All the experiments for each sample were performed in triplicate. The reaction mixture (Real Master Mix SYBR Green I; TIANGEN, China) contained 1 μL of cDNA, 10 μL of SYBR Green mix, 8 μL of ddH₂O, and 0.5 μL each of forward and reverse primers (10 μM). The PCR protocol included the following steps: 95 °C for 3 min (1 cycle) and 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s (40 cycles). The relative gene expression levels were normalized by comparison with the levels of GAPDH (Anas platyrhynchos). The 2^−ΔΔCt method was used to evaluate the relative gene expression levels. All the experiments were performed using CFX96TM Real-Time System (Bio Rad, Hercules, CA, USA).

The recombinant strains were cultured in MS medium with 1 % (W/V) glucose. RNAs were extracted from exponentially growing cells in baffled flasks using the RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing, China) as described by the manufacturer. The cDNA was amplified using FastQuant RT Kit (Tiangen, Beijing, China) with the total mRNA as the templates. Samples were then analyzed using a Light Cycler^®480 II (Roche, Basel, Switzerland) with RealMasterMix (SYBR Green I) (Tiangen, Beijing, China). Quantity real-time PCR amplification primers were listed in Additional file 1: Table S1; they exhibited identical calculated annealing temperatures and resulted in product sizes of approximately 50–200 bps. For data analysis, the rrsA gene was selected as internal standard for normalization between samples and three biological replicates were performed. The obtained data were analyzed by using the 2^−ΔΔCt method according to described previously [38 (link)].

At week 16, the mice (n = 6/group) were perfused intracardially with 4 °C PBS under anesthesia, after which WAT were quickly removed and stored in liquid nitrogen until RNA isolation. Total RNA was isolated using Trizol LS reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using a Quantscript RT Kit (TIANGEN Biotech, Beijing, China).
The cDNA was used to assess the relative expression of genes using RealMasterMix (SYBR green I) according to the manufacturer’s protocol (TIANGEN Biotech, Beijing, China). Real-time measurements of gene expression were performed with a DNA Engine Opticon 2 Real-Time Cycler PCR detection system (Bio-Rad Lab., Richmond, CA, USA). The primers used to measure gene expression are described in Table 1.

Semiquantitative real-time PCR (qRT-PCR) was performed on 200 ng of total RNA extracts that had been polyadenylated and reverse transcribed into cDNA using an anchored oligo (dT) primer (Tiangen, Beijing, China). The miRNA was transcribed into first-strand cDNA using miRcute miRNA first-strand cDNA synthesis kit (Tiangen, Beijing, China). The miRNA primers were listed in the supplementary Table 1A.
mRNA was transcribed into first-strand cDNA using Quantscript RT Kit (Tiangen, Beijing, China). qRT-PCR was run using the RealMasterMix (SYBR green I) (Tiangen, Beijing, China). The genes include Drosha, DRCG8, Dicer1 and the target genes of some miRNAs. RT-PCRs were run on the CFX96 Real-time PCR machine (BIO-RAD, Richmond, CA). The primers were marked in the supplementary Table 1B. Each RT reaction contained 11.25 uL 2.5 × RealMasterMix, 0.3 uL stem loop RT specific primer, 200 ng sample in 25 uL according to the Kit Introduction. Target sequences were amplified by being incubated at 94 °C for 2 min, followed by 40 cycles of 94 °C for 20s and 58 °C for 34s. β-actin and 5S RNA were used as endogenous normalization control for genes and miRNA separately. All assays were analyzed using the delta–delta–Ct method.

Quantitative real-time PCR (qRT-PCR) was performed on 200 ng of total RNA extracts that had been poly-adenylated and reverse transcribed into cDNA using an anchored oligo(dT) primer (Tiangen, Beijing, China). The miRNA was transcribed into first-strand cDNA using miRcute miRNA first-strand cDNA synthesis kit miRNA (Tiangen, Beijing, China). PCRs were run using the miRcute miRNA qPCR detection kit (Tiangen, Beijing, China). The miRNA primers were designed according to the instrution.
mRNA was transcribed into first-strand cDNA using Quantscript RT Kit (Tiangen, Beijing, China). qRT-PCR was run using the RealMasterMix (SYBR green I) (Tiangen, Beijing, China) with β-actin being the internal control. The genes include Drosha, DRCG8, Dicer1, Nrf2, Keap1 and the target genes of some miRNAs. qRT-PCRs were run on the ABI 7500 Real-time PCR machine (Applied Biosystems, Foster City, USA). Data were analyzed using the delta–delta–Ct method.