Taqman rna to cttm 1 step kit

HepG2 cells (3×10
⁵ cells/well) were treated with extract (0.25 or 1 µg/mL), compounds or controls (0.5 or 2 µM) for 0, 4, 6 or 8 hours. After treatment, mRNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Maryland, USA, Cat No. 74134). mRNA was quantified with a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA). The expression of the
HMOX-1 and
NQO1 genes
^{21 (link),
39 (link)–
41 (link)} was evaluated by RT-qPCR using predesigned TaqMan Gene Expression Assays (Hs01110250_m1 and Hs01045993_g1) and the TaqMan® RNA-to-CT
^TM 1-Step Kit (Applied Biosystems, Foster City, CA, Cat No. 4331182). The run method was holding at 48°C for 15 min, 95°C for 10 min and cycling (40 cycles) at 95°C for 15 sec and 60°C for 1 min. β-actin (Applied Biosystems, Foster City, CA, ref. 4325788) was used as an endogenous control.

Quantification of mRNA expression levels was measured according to a previously established method [11 (link),12 (link),13 (link)]. Extraction of total RNA from cells was performed using QIAshredder and RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Real-time PCR was performed using a TaqMan^® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) and TaqMan^® RNA-to-CT^TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA). The assay IDs of the TaqMan probes used were: CTL1, Hs00223114_m1; CTL2, Hs01105936_m1; SMPD1, Hs03679345_m1; SMPD2, Hs00906924_g1; SMPD3, Hs00920354_m1; SMPD4, Hs004187047_g1; SMPDL3A, Hs01041066_m1; SMPDL3B, Hs01038741_m1; survivin, Hs00977612_mL; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs99999905_mL; human β-actin, Hs99999903_mL; mouse CTL1, Mm01350815_m1; and mouse β-actin, Mm00607939_s1. RT-PCR was performed using the LightCycler^® 96 system (Roche Diagnostics, Mannheim, Germany). Relative expression was calculated as the expression level of the target gene relative to the housekeeping gene using the ΔCt method [13 (link),15 (link),16 (link)].

Two weeks after germination, “Antero” (Wsm2-, WSMV susceptible) and “Snowmass” (Wsm2+, WSMV resistant) plants were subjected to WSMV inoculation as described above. Leaf samples were taken by cutting the whole leaf tissues from five biological replicates over the time course of 0-, 5-, 10-, and 15-dpi. Leaf tissues were ground and homogenized in liquid nitrogen for subsequent total RNA isolation with the Spectrum^TM Plant Total RNA Isolation Kit (Sigma, USA), followed by on-column DNase I digestion treatment (Sigma-Aldrich, USA) to remove genomic DNA according to the manufacturer's instructions. The one-step qRT-PCR reaction was carried out using the TaqMan® RNA-to-Ct^TM 1-step kit (Applied Biosystems^TM, USA) on a QuantStudio^TM3 Real-Time PCR system (Applied Biosystems, USA). Reaction conditions and parameters adopted were as described previously by Price et al. (2010 (link)). WSMV was detected via qRT-PCR using coat protein-specific primers and the probe listed in Supplementary Table S3. To quantify the absolute amount of WSMV coat protein transcript levels, a regression line was plotted using plant RNA that contains 10 ng/μL of WSMV and subjected to 10-fold serial dilutions to 1 × 10⁻⁵ ng/μL. The C_q values for each dilution were plotted against total RNA transcript levels, and the regression line was considered at R² > 0.996.

RT-PCR analysis was performed as previously described [8 (link),9 (link),10 (link)]. MCF10A, MIA PaCa-2, and PANC-1 cells were homogenized with QIAshredder (Qiagen, Venlo, The Netherlands), and total RNA was purified using the RNeasy^® Mini Kit (Qiagen, Venlo, The Netherlands). TaqMan probes for the target mRNAs were designed using TaqMan^® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Assay IDs of the TaqMan probes used were: CHT1, Hs00222361_m1; CTL1, Hs00223114_m1; CTL2, Hs01105936_m1; CTL3, Hs00537043_m1; CTL4, Hs00228901_m1; CTL5, Hs01120485_m1; OCT1, Hs00427552_m1; OCT2, Hs01010726_m1; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs02786624_m1 [8 (link),9 (link),10 (link)]. Real-time PCR was measured using the TaqMan^® RNA-to-CT^TM 1-Step Kit (Applied Biosystems, Foster City, CA, USA), and data analysis was performed using a LightCycler^® 96 system (Roche Diagnostics, Mannheim, Germany). The mRNA expression level relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the housekeeping gene, for each target gene was calculated according to the comparative threshold-cycle (C_t) method [8 (link),9 (link),10 (link)].