E z n a total rna kit 2

To perform the analysis, cells were collected and Total RNA was isolated with the E.Z.N.A.® Total RNA Kit II (Omega Bio-tek) according to the manufacturer’s protocol. cDNAs were synthesized using PrimeScript™ RT Master Mix (Perfect Real Time) (RR036A, Takara) as previously described [22 (link)]. Quantitative PCR analysis was performed using the TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (RR820A, Takara) as previously described [22 (link)]. To measure mRNA stability, cells were treated with Actinomycin D (5 μg/ml), and total RNA was extracted at the indicated times and subjected to q-PCR analysis.
Human CNOT3 forward: 5′-GGACGTTCCACAGACAGTGA-3′; reverse: 5′-GAGGGTGCTGGTTGCTGT-3′.
Human c-Jun forward: 5′-TCCAAGTGCCGAAAAAGGAAG-3′; reverse: 5′-CGAGTTCTGAGCTTTCAAGGT-3′.
Human TSC1 forward: 5′-CAACAGGCGTCTTGGTGTTG-3′; reverse: 5′-ACACACTGGCATGGAGATGG-3′.
Human GAPDH forward: 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse: 5′-TGGTGAAGACGCCAGTGGA-3′.

Total RNA isolation was achieved using E.Z.N.A.^® Total RNA Kit II (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer’s instructions. Generation of cDNA was performed as previously described [19 (link)]. For real-time PCR, LightCycler^® 480 II devices (Roche, Basel, Switzerland) were used with forward and reverse primers from Sigma-Aldrich. Primer sequences can be found in Table 1.

Total RNA was extracted using an E.Z.N.A.® Total RNA Kit II (Omega Bio-Tek, Norcross, GA) and reversely transcribed into cDNA using a ReverTra Ace-a Kit (Toyobo, Osaka, Japan). SYBR-Green real-time RT-PCR was performed using the Stratagene Mx3005P sequence detection system (Agilent Technologies, Santa Clara, CA) and SYBR Premix EX Taq II (2×) (Takara, Shiga, Japan). GAPDH mRNA was used to normalize the RNA inputs. All primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

Total RNA was extracted using the EZNA Total RNA Kit II (OMEGA Biotek, Inc., GA, USA) according to the manufacturer’s instructions. Extracted total RNA was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Shiga, Japan) as instructed. qRT-PCR analysis was performed using the SYBR Premix Ex Taq II Kit (TaKaRa), and the results were assessed with a CFX96 Real-time RT-PCR System (Bio-Rad, Hercules, CA, USA). β-Actin was used as the endogenous control to normalize the expression levels. The resulting amplification and melt curves were analyzed to ensure the identity of the specific PCR product. Threshold cycle values were used to calculate the fold change in the transcript levels by using the 2^−ΔΔCt method. The primers used for qRT-PCR were as follows: β-actin: sense 5′-TGGCACCCAGCACAATGAA-3′ and antisense 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′; p53: sense 5′-CAGCACATGACGGAGGTTGT-3′ and antisense 5′-TCATCCAAATACTCCACACGC-3′; p21: sense 5′-TGTCCGTCAGAACCCATGC-3′ and antisense 5′-AAAGTCGAAGTTCCATCGCTC-3′; and p16: sense 5′-GAAGAAAGAGGAGGGGCTG-3′ and antisense 5′-GCGCTACCTGATTCCAATTC-3′.

Mice were euthanized at the end of 4-week feeding. Three of five mice from each dietary group with greatest, medium, and least fat mass were used for RNA-Seq in the current study. In order to be consistent, right side of BAT at the interscapular region was collected, weighed, flash frozen, and stored at −80 °C. Total RNA was isolated for sequencing using E.Z.N.A^® Total RNA kit II (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. Briefly, ~20 mg BAT were homogenized in 1 mL of RNA-solv reagent using bullet blender^® tissue homogenizer (Next Advance Inc., Averill Park, NY, USA). The homogenized tissues were then processed with chloroform addition and phase separation. The aqueous phase was transferred to a new sterile 1.5 mL tube and mixed with equal volume of 70% ethanol. The mixtures were loaded to the Hibind^® RNA spin column and processed according to the manufacturer’s instruction. Total RNA was eluted in 30 μL nuclease-free water and stored in −80 °C. The concentration and purity of extracted RNA samples were checked using a NanoDrop^TM spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), then analyzed using an Agilent RNA 6000 pico kit with an Agilent’s 2100 Bioanalyzer (Agilent Technologies Inc., Waldbronn, Germany). High quality of RNA samples with an RNA integrity number (RIN) above 6.5 were used for analysis.

Total RNA was isolated from RAW264.7 cells and BMDMs using the EZNA Total RNA Kit II (Omega, Bio-Tek, Norcross GA, USA, Cat#: R6934-01). MonScript RTIII All-in-One Mix with dsDNase (Monad Biotech Co., Ltd, Shanghai, China, Cat#: MR05101 M) was used to reverse transcribe the RNA, and real-time PCR was performed using MonAmp ChemoHS qPCR Mix (Monad Biotech Co., Ltd, Shanghai, China, Cat#: MQ00401S) on an Mx3005p real-time PCR instrument. For real-time PCR, sequence-specific primers for vimentin, α-SMA, and GAPDH were as follows: vimentin: forward 5′-GCAGTATGAAAGCGTGGCTG-3′, reverse 5′-CTCCAGGGACTCGTTAGTGC-3′; α-SMA: forward 5′-TCAGGGAGTAATGGTTGGAATG-3′, reverse 5′-CCAGAGTCCAGCACAATACCAG-3′; GAPDH: forward 5′-CCTCGTCCCGTAGACAAAATG-3′, reverse 5′-TGAGGTCAATGAAGGGGTCGT-3′. The mRNA levels of α-SMA, vimentin, and GAPDH were used as housekeeping genes for normalization and were calculated using the 2^−ΔΔCT method.

RNA extraction on GN11 and FNCB4 cell lines was performed using the EZNA Total RNA Kit II (Omega Bio-Tek, R6934-02), and the cDNA was reverse-transcribed using the SuperScript III kit (Thermo Fisher Scientific, 18080051). qPCR was carried out on Applied Biosystems 7900HT FasT Real-Time PCR System using exon span–specific TaqMan Gene Expression Assay (Applied Biosystems, 4440040). The following primers were used: Jag1 (Mm00496202_m1), Notch1 (Mm00627185_m1), Notch2 (Mm00203077_m1), Notch3 (Mm1345646_m1), Notch4 (Mm00440525_m1), JAG1 (Hs01070032_m1), NOTCH1 (Hs01062014_m1), NOTCH2 (Hs01050702_m1), NOTCH3 (Hs01128537_m1), NOTCH4 (Hs00965889_m1), and 18S (Hs99999901_s1) and Actb (Mm00607939) as housekeeping genes. Amperase activation was achieved by heating at 50°C for 2 minutes, before denaturation at 95°C for 20 seconds, followed by 40 cycles of 1 second at 95°C with 20 seconds of extension time at 60°C. Gene expression data were analyzed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied Biosystems). Results were analyzed with the standard ΔC_T method and normalized to the expression of the housekeeping genes.