Taqman probe based gene expression assay

Total RNA was isolated from E11.5 mandibular pharyngeal arches using the High Pure RNA Isolation Kit (Roche). This RNA was then used to synthesize cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For qRT-PCR, cDNA was amplified using TaqMan Probe-Based Gene Expression Assays (Applied Biosystems) and the QuantStudio 3 Real-Time PCR System (ThermoFisher). Normalization to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)was used to determine relative gene expression and statistical analysis automatically applied by the instrumental software. Significance of qRT-PCR results were determined by a two-tailed students t-test followed by post hoc Benjamini-Hochberg FDR correction as automatically calculated by the QuantStudio 3 qRT-PCR thermal cycler software analysis package.

Total RNA was isolated from huPCLS and qPCR analysis was performed as previously described [15 (link),25 (link),36 (link)] using individual TaqMan^® Probe-Based Gene Expression Assays (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) selected for proinflammatory cytokines (IL-1β, IL-6, TNFα, and IL-1α), inflammation (COX-2), relevant cytoprotective and antioxidant enzymes (heme oxygenase-1 (HMOX1) and NADPH:quinone oxidoreductase-1 (NQO1), and cell cycle markers (TP53, CDK2, CDKN1A, CDKN2A, CDK4, CDK6, RB, and E2F). Transcript levels of tested genes were normalized to β-Actin.

Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA). One μg of total RNA was reverse-transcribed with ReverTra Ace qPCR RT Master Mix (Toyobo, Japan). For quantitative mRNA expression analysis, TaqMan RT-PCR was performed on the StepOnePlus™Real-Time PCR system following the manufacturer's instructions. TaqMan Probe-Based Gene Expression Assays were used for IL-6; Hs 00174131_m1 and IL-6R; Hs 01075667_m1 (Applied Biosystems). GAPDH was used as an internal control. Relative levels of mRNA gene expression were calculated using the 2^-ΔΔCT method as described previously.[21 (link)]

Quantitative polymerase chain reaction (qPCR) was performed as previously described [22 (link)]. Briefly, TaqMan^® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA) were used. To evaluate the effect of SDG treatment on the mRNA expression of antioxidant genes, individual TaqMan^® gene expression assays were selected for antioxidant enzymes (heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase, quinone 1 (NQO1), and glutathione S-transferase mu 1 (GSTM1)).
Briefly, cells were pre-treated with SDG (50 μM, 6 h) and irradiated (2 Gy). Total RNA was isolated from using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti^® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). qPCR was performed using 25 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA and calibrated to untreated control samples according to the ΔΔC_T method as shown previously [22 (link)].

Quantitative Polymerase Chain Reaction (qPCR) was performed using TaqMan^® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Individual TaqMan gene expression assays were selected for GADD45α, Survivin (Birc5: baculoviral inhibitor of apoptosis repeat-containing 5), and Bcl-2-associated X protein (BAX). Briefly, cells were lysed and RNA was isolated using a commercially available kit, QIAprep Spin Miniprep Kit, supplied by Qiagen (Valencia, CA, USA). Total RNA was quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti^® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Quantitative real-time PCR was performed using 50 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA housekeeping gene and calibrated to the control samples according to the 2^−ΔΔCt method as previously described [40 (link),49 (link),50 (link)].