Sybr green master mix

Real-time quantitative reverse transcription-PCR (qRT-PCR) was applied to verify the RNA-Seq results. Total RNA was isolated from leaves of Huiyuan treated by different lights and then used to generate cDNA. cDNA was synthesized by using an EASYspin Plus Plant RNA Kit (Aidlab Biotechnologies Co., Ltd.; Beijing China) with gDNA Eraser (Takara). Each reaction (final volume, 10 μL) contained 5 μL 2 × SYBR Green master mix (Roche), 0.5 μL of each the forward and reverse primer (10 μM), 1 μL of the cDNA template, and 3 μL of RNase free water. qRT-PCR was conducted in a LightCycler 480 system (Roche, United States) under the following parameters: 95°C for 5 min and 45 cycles of 95°C for 10 s, 58°C for 15 s, and 72°C for 20 s. The gene expression levels were calculated with the 2^–ΔΔT method. The cotton GhUBQ7 gene (DQ116441) was amplified as an internal control gene. For each tissue, three biological replicates each with three technical replicates were analyzed. The list of primers was shown in Supplementary Table 1.

Samples identified as non- Rattus norvegicus using the RFLP-method were subjected to further PCR and sequencing of the mitochondrial cytochrome b (cytb) gene to discriminate between closely related Rattus species. The PCR and sequencing of cytb gene were performed using primers mcytbHb (5’-GAATGGGAGAATGAAGTGGAATGCG-3’) and mcytb1 (5’-CCATCGTTGTAATTCAACTATAG-3’)^{8 (link)}. Each PCR reaction contained 1.5 µl of a prepared primer mix (with final concentrations of each primer being 2.5 µM), 1.5 µl extracted DNA from kidneys, 15 µl 2 × SYBR green master mix (Roche, Switzerland) and water to a final volume of 30 µl. The PCR reactions were performed and analysed using LightCycler 2.0 (Roche, Switzerland), with an initial holding temperature of 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min, and 1 cycle of 10 min at 72 °C. Obtained PCR amplicons were purified using QIAquick PCR Purification Kit (QIAGEN, Germany) and sequenced using both the PCR amplification primers. The resulting DNA sequencing chromatogram were assembled using SeqMan Pro (DNASTAR Lasergene, USA). The sequences derived was aligned against the cytb gene sequence entries in GenBank using the online BLAST search engine of the National Center for Biotechnology Information (NCBI) to detect sequences with high similarity for species identification.

Quantitative real time PCR was performed on total RNA extracted with a mirVana’s isolation kit (Ambion, Austin, TX, USA) from the frontal cortex of human samples. Purified RNAs were used to generate the corresponding cDNAs, which served as PCR templates for mRNA quantification. PCR amplification and detection were performed with the ROCHE LightCycler 480 detector, using 2× SYBR GREEN Master Mix (Roche, Basel, Switzerland) as reagent, following the manufacturer’s instructions. The reaction profile was: denaturation–activation cycle (95 °C for 10 min) followed by 40 cycles of denaturation–annealing–extension (95 °C for 10 min, 72 °C for 1 min, 98 °C continuous). mRNA levels were calculated using the Light Cycler 480 software (Roche). Data were analyzed with SDS v. 1.9.1 Software (Applied Biosystems, Madrid, Spain) following the 2^−ΔΔCT method of Applied Biosystems. Primers were as follows: RELN (5’-actctgtcaacagctcaagc-3’) and (3’-tggtcaattgcccagctttg-5). The results were normalized for the expression levels of the housekeeping gene, GAPDH (5’-tccaaaatcaagtggggcga -3’ and 3’-tctccatggtggtgaagacg -5’), which were quantified simultaneously with the target gene in each sample.

Total RNA was extracted from brains or cells using TRIzol according to the manufacturer’s instructions. Next, the mixed genomic DNA was digested with DNase I and 1-μg RNA was reverse transcribed using the reverse transcriptase (TaKaRa Biotechnology, Dalian, China) from avian myeloblastosis virus with oligonucleotide (dT) 18. cDNA (0.5 μl) was used as the template for qRT-PCR. In addition, the reaction mixtures contained 5-μl 2× SYBR green master mix (Roche, Indianapolis, IN, USA), 0.25 μl of each primer (10 mM), and 4-μl ultrapure water. The assay was run on an ABIViiA 7 PCR system (Applied Biosystems, Waltham, MA, USA). The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control.

Total DNA was isolated from sWAT with QIAamp DNA Mini Kit (no. 51306; Qiagen, Hilden, Germany), following the provided manufacturer’s protocol. The PCR reaction was performed in a LightCycler 480 (Roche). The reaction mix contained 500 pg of DNA, 1.25 µM of forward and reverse primer, and 4 µL of 2× SYBR green master mix (Roche) in a final volume of 8 µL. Then, 16S and Cox2 were used as mitochondrial-encoded genes, and Hk2 and Ucp2 were used as nuclear-encoded genes. Primer sequences are listed in Table 2. Data were analyzed with LightCycler software release 1.5.0 (Roche). The mean PCR efficiency was calculated to assess gene expression levels using LinRegPCR program version 12.17 [40 (link)].