Cdna archive kit

HUVECs or ECFCs (8000 cells/well) were plated in collagen I coated 96-well plates. Medium was removed 16 hours later, and BMP or vehicle was added in media with 100 μg/ml BSA. RNA was harvested 24 hours later via Qiagen RNeasy kit including the DNAse treatment step per manufacturer’s instructions. RNA (500 ng) was converted to cDNA using Life Technologies cDNA archive kit per manufacturer’s instructions. Quantitative PCR analysis was performed with the following Applied Biosystems (Grand Island, NY) Taqman primer/probes: c-kit Hs00174029_m1, FGFR1 Hs00915142_m1, VEGFR2 Hs00911710, Kit ligand Hs00295067_s1, MCP-1 Hs00234140_m1, MMP-1 Hs00899658_m1, PECAM-1 Hs00169777_m1, PlGF Hs01119262_m1, β-actin 4333762T, GAPDH 4326317E, PGK1 5333765T, and PPIA 4333763T.

The relative quantification of mRNA was performed with a quantitative RT-PCR assay. 400 ng of total RNA was transcribed into cDNA using the cDNA Archive Kit (Life Technologies, Foster City, CA, USA). Each cDNA sample was analyzed by quantitative real-time PCR (qPCR) using the fluorescent TaqMan 5’-nuclease assays (Life Technologies). The analysis was performed with ViiA 7 detection system and software (Life Technologies). Gene expression was standardized to POLR2A expression (2^-ΔCt).
miRNAs were reverse transcribed using TaqMan Micro RNA Reverse Transcription Kit (Life Technologies). Taqman microRNA assays (Life Technologies) were used for miRNA quantification.

Gene expression in BMDCs was analyzed by real-time RT-PCR using TaqMan probes. Briefly, RNA was extracted using Zymo quick kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocols. Complementary DNA was synthesized using the cDNA archive kit (Life Technologies, Carlsbad, CA, USA). TaqMan primers and probes for both mouse and human genes (Irf7—Mm00516788_m1, IRF7—Hs01014809_g1; Isg15—Mm01705338_s1, ISG15-Hs01921425_s1, Ifnβ—Mm00439546_s1, and Cxcl10—Mm00445235, CXCL10-Hs00171042_m1) were purchased from Applied Biosystems (Foster City, CA, USA). Cyclophilin (Mouse—Mm02342430_g1) was used as the reference gene for normalization for mouse cells and GAPDH (Human—4310884E) was used for human cells. The primer sequences are available from the Applied Biosystems website. The cycle threshold (Ct) method of relative quantification of gene expression was used (ddCt), and the normalized Ct values (against cyclophilin or GAPDH for mouse and human cells, respectively) were calibrated against the control (untreated cells) in each experiment. Percent fold change was calculated by normalizing the values to the CpG, R848, or IFN-α treatments alone in each experiment. Mean and SEM were obtained from at least three independent experiments from independent cultures from different mice or different human donors, and are presented in the graphs.

RNA was reverse transcribed using the high-capacity complementary DNA (cDNA) archive kit (Life Technologies). qPCR was performed using the StepOnePlus™ Real-Time PCR systems (Applied Biosystems) (23 (link)). The primer-probe sets for CYP11B2 were designed in house and manufactured by IDT DNA (24 (link)). The following primer-probe sets were purchased from Thermo Fisher Scientific: LHCGR (Hs00174885_m1), GNRHR (gonadotropin-releasing hormone receptor) (Hs00171248_m1), and ACTB (β-actin) (Hs01060665_g1). ACTB transcript was used as an internal control for quantitative normalization. The delta-delta threshold cycle method was used to calculate fold changes in mRNA expression over adjacent normal adrenal.
This study was approved by the institutional review boards at the National Hospital Organization Kyoto Medical Center (20–038) and the University of Michigan (HUM00083056). The patient provided written consent for the use of specimen in this study and publication of this article.

Gene expression in cDCs was analyzed by real-time quantitative reverse transcriptase-PCR (qRT-PCR) using TaqMan probes as described before [44 (link), 45 (link)]. Briefly, RNA was extracted using Qiagen RNasy plus kit (Qiagen Inc., Valencia, CA, USA), following the manufacturer's protocols. cDNA was synthesized using the cDNA archive kit (Life Technologies, Grand Island, NY, USA). TaqMan primers and probes for TLR4, TLR9, and ERalpha were purchased from Applied Biosystem. Cyclophilin was used as the reference gene for normalization. The Ct method of relative quantification of gene expression was used for these TaqMan PCRs (ΔΔCt), and the normalized Ct values (against cyclophilin) were calibrated against the control sample in each experiment.

Gene expression in brain, kidney, and spleen samples was analyzed using TaqMan probes and real time quantitative PCR (qPCR). RNA was extracted using TriZol (Invitrogen) following the manufacturers protocol. DNA removal was performed using DNA-free^TM Kit (Invitrogen) following the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized with the cDNA archive kit (Life Technologies, CA, United States). qPCR was performed using TaqMan primers and probes for mouse genes Irf7 (Mm00516788_m1), Cxcl10 (Mm00445235), ISG15 (Mm01705338_s1), and ACE (Mm00802048_m1; Applied Biosystems, CA, United States). Cyclophilin was used as reference gene (Mm02342430_g1). ΔΔCt method was used to calculate the fold changes in gene expression by normalizing the values to the control group in each experiment.