Revertra ace qpcr master mix

Based on the results of a previous study.^{17 (link)}, we selected ZmCyst, ZmAOS, and ZmTPS10 as the genes to be monitored. After the compound treatment, the tips (5 cm from the top) of the primary leaves of maize seedlings were collected and RNA was extracted using a FavorPrep Plant Total RNA Purification Mini Kit (Favorgen, Ping Tung, Taiwan). After removing the DNA with a DNA-free DNA Removal Kit (Thermo Fisher Scientific, Waltham, MA), a portion of RNA (1 µg) was used to synthesize cDNA using ReverTra Ace qPCR Master Mix (Toyobo, Osaka, Japan). Quantitative reverse transcription PCR (RT-qPCR) was performed using a StePOnePlus System (Thermo Fisher Scientific) with SYBR Green Master Mix (Thermo Fisher Scientific). Primers and gene IDs are listed in Supplemental Table S1. The maize adenine phosphoribosyl transferase (ZmAPT1) gene was used as an internal standard to normalize cDNA concentration, and the relative expression levels of the target genes were obtained.

Total RNA was isolated from sorted NPCs with the use of RNAiso plus (Takara), and up to 0.5 μg of the RNA was subjected to RT with the use of ReverTra Ace qPCR Master Mix (Toyobo). The resulting cDNA was subjected to real-time PCR analysis in a LightCycler 480 instrument (Roche) with any of KAPA SYBR FAST for LightCycler 480 (Kapa Biosystems), Thunderbird SYBR qPCR mix (Takara), and QuantiNova SYBR Green PCR kit (Qiagen). The amount of target mRNA was normalized by that of β-actin mRNA. The sequences of PCR primers are provided in Supplementary Table 1.

BMDMs were stimulated with CpG (0.063 μg/ml), IFN-γ (100 U/ml) + αIL-10R (1.25 μg/ml), or with all three agents at 37 °C for 20 h. Total RNA from each BMDM sample was then extracted using RNeasy Plus Mini Kits (QIAGEN) according to the manufacturer's instructions. Microarray analyses were performed by MBL, Japan, using SurePrint G3 Mouse Gene Expression 8 × 60 K Microarrays (Agilent). For qPCR analyses, RNA was reverse transcribed using ReverTra Ace qPCR master mix (TOYOBO, Japan), and cDNA products were amplified using a LightCycler 96 (Roche) with Universal SYBR Select master mix (Thermofisher). Data were analyzed using the delta Ct method and were normalized to β-actin RNA expression in each sample. The original microarray data were deposited in GEO database under accession number GSE90881. The primers for real-time PCR were as follows: β-actin-Fw, 5′-CTTTGCAGCTCCTTCGTTG-3′ and β-actin-Rv, 5′-CGATGGAGGGGAATACAGC-3′; ICAM-1-Fw, 5′-GTCCGCTGTGCTTTGAGAAC-3′ and ICAM-1-Rv, 5′-TGAGGTCCTTGCCTACTTGC-3′; VCAM-1-Fw, 5′-GAAATGCCACCCTCACCTTA-3′ and VCAM-1-Rv, 5′-CGGGGGAGATGTCAACAATA-3′; LFA-1α-Fw, 5′-CAGAACAAGAACCCCGATGT-3′ and LFA-1α-Rv, 5′-CCTGGCACCAGACTCTTCTT-3′; and VLA-4α-Fw, 5′-AATGCCTCAGTGGTCAATCC-3′ and VLA-4α-Rv, 5′- TCTCCTCCAGGCATGTCTTC-3′.