Cfx384 c1000 thermal cycler

Total RNA was extracted from the human eSCs using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States), and 2 μg of this RNA was used to synthesize cDNA using the TOPscript^TM RT DryMIX kit (dT18 plus) following the manufacturer’s instructions (Enzynomics, Daejeon, South Korea). Quantitative reverse transcription PCR (qRT-PCR) was conducted using TOPreal^TM qPCR 2X PreMIX (SYBR Green with high ROX) (Enzynomics) and a CFX384 C1000 Thermal Cycler (Bio-Rad, Hercules, CA, United States). Human L19 was used to normalize IL-8 expression, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize the expression of the other genes. The primers used for qRT-PCR, such as for FOXO1, GAPDH, IGFBP1, PRL (Baek et al., 2018 (link)), L19 (Francis et al., 2006 (link)), p16 (Zhang et al., 2017 (link)), and p53 (Zhou et al., 2017 (link)) have been previously reported. Primers for IL8 and p21 are listed in Supplementary Table 4.

Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND2000, Thermo Scientific). qPCR were performed with a CFX384 C1000 thermal cycler (Bio-Rad) using the Super Scrip III Platinum One Step qRT-PCR kit (Invitrogen) and TaqMan probes (Applied Biosystems): 4326317E (GAPDH), Hs00173499_m1 (S1P1), AJ39RQ5 (S1P2), Hs00245464_s1 (S1P3), Hs02330084_s1 (S1P4) and Hs00928195_s1 (S1P5) according manufacturer’s instructions. Samples were measured as quadruplicates. The relative expression of each gene was calculated according to the ΔΔCT method [22 (link)]. Expression of the housekeeping gene GAPDH was used to normalize for variations in RNA input.

Aqueous phase containing total RNA was isolated from human and mouse brain regions or cells using TRI Reagent (Sigma-Aldrich) following the manufacturer’s protocol. Then total RNA was purified with Direct-zol RNA MiniPrep Plus (Zymo Research, Irvine, CA, United States). A range of 0.25 to 1 μg of RNA for each condition was reverse transcribed using a PrimeScript RT reagent kit (Takara, Japan). The RT reaction was performed at 42°C for 60 min followed by an additional 5 min at 70°C. cDNA was diluted to 5 ng/μL and 2 μL was used to perform qRT-PCR. PrimeTime qPCR assays (Table 2) were used as recommended by provider (IDT technologies, United States). qRT-PCR was carried out with Premix Ex Taq (RR390A, Takara, Japan) in 6 μL final volume using a CFX384-C1000 thermal cycler (Bio-Rad Laboratories, Madrid, Spain). The two-step amplification program was: 40 cycles of 2 min at 95°C for denaturation and polymerase activation, 5 s at 95°C for denaturation, and a final extension for 20 s at 60°C. For each target gene, the expression level was determined using a standard curve (efficiency between 93 and 100%) and normalized to housekeeper gene mRNA levels. Reactions were performed in triplicate to reduce variability. The ΔΔCt method was used to analyze the data.

To RNA, a mastermix made of 2ROX, Taq‐polymerase (Superscript^™ III RT Platinum^® Taq‐mix 1325133; Invitrogen), probes for apoM (Hs01597780_g1), apoA1 (Hs0098500_g1), apoE (Rh0279929_m1), albumin (Rh02828765_m1), Sgpp1 (Mf04372495_m1), Sgpp2 (Rh00544786_m1), S1PL (Rh00393705_m1), Sphk1 (Hs00184211_m1), Sphk2 (Rh02876562_m1), serum amyloid A (SAA) (Hs00293702_m1) and the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, Rh02621745_g1), (Applied Biosystem, Foster City, CA, USA) were added. qRT‐PCR reactions were performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) or CFX384 C1000 Thermal cycler (Bio Rad, USA). Changes in mRNA levels were calculated according to the 2^−ΔΔC_T method 36.

Gene expression levels of mRNAs and long non-coding RNAs (lncRNAs) shown previously to be regulators of beta-cell death and cellular impairment (13 (link), 18 (link), 19 ) were investigated by real-time quantitative PCR (qPCR). RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Real-time qPCR was carried out with TaqMan Assays and TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA) using a CFX384 C1000 Thermal cycler (Bio-Rad). Relative expression levels were analyzed using the 2^-ΔΔCt method (20 (link)) with normalization to GAPDH. GAPDH was chosen as this was the most stable housekeeping gene measured in EndoC-βH5 cells by real-time qPCR among GAPDH, Peptidylprolyl isomerase A (PPIA), hypoxanthine Phosphoribosyltransferase 1 (HPRT1), beta-actin (ACTB) and 18S ribosomal RNA (data not shown).