Primer 5

In order to be more representative of the chip as a whole, 40 sets of BC and ANNB tissues were randomly selected from the microarray for further verification by RT-qPCR analysis. Total RNA was extracted from each frozen sample (the amount of tissue was ~100 mg) in accordance with the manufacturer's protocol (RNAiso PLUS; Takara Bio, Inc.). The 1% agarose electrophoresis determined the integrity of the isolated RNA, and PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc.) was used to synthesize first-strand cDNA. A spectrophotometer was used to measure the optical density ratio at 260 and 280 nm (OD260/280=1.8–2.0) to determine RNA concentration. Primer 5.0 software (Premier Biosoft) was used to design specific primers, which are listed in Table SI. A StepOnePlus real-time PCR system with SYBR-Green I (Takara Bio, Inc.) was used to conduct RT-qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Each sample was tested in triplicate. The thermocycling conditions used were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. Relative mRNA expression levels were determined using the 2^−ΔΔCq method (21 (link)).

In accordance with the manufacturer’s instructions, total RNA from all the
hippocampal tissues was extracted using Trizol reagent (Invitrogen, Carlsbad,
CA, USA). A reverse-transcription reaction was conducted to generate cDNA
(PrimeScript® RT reagent kit, Takara Biotechnology [Dalian] Ltd., Dalian,
China). The primers, which were designed using Primer 5.0 software (PREMIER
Biosoft International, San Francisco, CA, USA), were as follows:
Mouse PPARγ, Forward: 5ʹ-GAGTAGCCTGGGCTGCTTTT-3′;
Reverse: 5ʹ-ATAATAAGGCGGGGACGCAG-3′;
Mouse β-actin, Forward: 5ʹ-AGAGGGAAATCGTGCGTGAC-3′;
Reverse: 5ʹ-CAGGAAGGAAGGCTGGAAG-3′.
Quantitative real-time reverse-transcription polymerase chain reactions
(qRT-PCRs) with SYBR Green detection for PPARγ and β-actin gene expression were
used (SYBR® Premix Ex Taq™ II, Takara Biotechnology [Dalian] Ltd.) on an ABI
7500 RT-PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The Ct method
was used to analyze the results by calculating 2-ΔΔCt using the following
formula: ΔCt = target gene Ct value −glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) Ct value and ΔΔCt = ΔCt treatment − ΔCt control.

Eight blood samples were randomly taken from each of the treatment groups of both broiler breeds. The corresponding wing number (from which the blood sample was collected) was recorded. RNA was extracted from each sample. The quantified RNA solution was stored at –80°C. The cDNA was prepared by reverse transcription using PrimeScript® RT reagent Kit with gDNA Eraser (Takara Biomedical Technology (Beijing) Co., Ltd.). A pair of primers (Table 1) was designed based on the HSP70 cDNA sequences (accession number: J02579) in GenBank (http://www.ncbi.nlm.nih.gov/) using Primer 5.0 software (Premier Biosoft, Palo Alto, CA). The amplification of HSP70 gene was performed and a 259-bp product was obtained. The expression level was determined by a 2-step quantitative real-time PCR (RT-qPCR). The reaction mixture was prepared in 96-well real-time PCR plates and consisted of 1 μL of cDNA, 0.4 μL of the upstream and downstream primers (20 μM), 10 μL of SYBR® Green Realtime PCR Master Mix, and 8.6 μL of ddH₂O. Each sample was analyzed in triplicate. If the Ct values of the same sample differed by more than 0.04, 2 additional reactions were performed. The cycling conditions used for RT-qPCR were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 20 s. A melting curve analysis was performed over a temperature range of 65 to 95°C (in increments of 0.5°C).