High capacity complementary dna cdna reverse transcription kit

Total RNA was extracted from intestinal-mucosal tissues using an RNeasy kit (QIAGEN) per the manufacturer’s protocol. We quantified RNA using a NanoDrop spectrophotometer (Applied Biosystems). A High Capacity Complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems) was employed for reverse transcription using 2 μg RNA. Subsequently, we performed real-time PCR reactions using a ViiA 7 Real-Time PCR System with PowerUp SYBR Green Master Mix Kit (both, Applied Biosystems). PCR reactions were incubated in a 384-well plate at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. All samples were assayed in triplicate, and data were normalized to endogenous control β-actin. We calculated relative RNA expression levels using the ^ΔΔCt method. Primers, which were modified from previous studies^{40 (link)–42 (link)} and synthesized by Invitrogen (Shanghai, China), were as follows in Supplementary Table 1.

Gene expression assay validation was performed using TaqMan® (Applied Biosystems, CA). A calibration curve was constructed using the GSH exposure (50 μg/ml); GSH is a positive inductor of the target gene, ATP-binding cassette C subfamily (ABCC1–4) [38 (link)–41 (link)]. Cells were exposed to PM_2.5 extract (25 μg/ml), BUD (0.05 μg/ml), and the Co-T at different time points (5, 6, and 7 hr). Total RNA was extracted using TRIZOL reagent (Invitrogen, CA). The high-capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems, CA) was used to synthesize cDNA. Quantitative florescent amplification of cDNA of ABCC1 (Hs01561502_m1), ABCC3 (Hs00978473_ml), and ABCC4 (Hs00988717_m1) was performed using TaqMan Gene Expression Assays (Applied Biosystems, CA). The real-time polymerase chain reaction (RTPCR) was conducted in a StepOne Real-Time PCR System (Applied Biosystems, CA). β-Actin (Hs03023943_ml) was used as a housekeeping gene to normalize the target genes.

Reverse transcription–quantitative real time PCR was performed to validate the relative expression of the most significant differentially expressed genes in transcriptome microarrays and reference genes (Table S4). This step was performed using a LightCycler^®480 II system (Roche, Basel, Switzerland). Reverse transcription reactions were performed using 500 ng (cells and tissue samples) and 150ng (exosomes samples) of total RNA, random hexanucleotides, and the High-Capacity cDNA (complementary DNA) Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), following the manufacturer’s instructions. RT-qPCR was performed with assays based on hydrolysis probes using 1 μL of cDNA, 2.5 μL of TaqMan Gene Expression Master Mix and 0.25 μL of TaqMan Gene Expression Assay (Applied Biosystems, Waltham, MA, USA) to a 5 μL final reaction volume. To calculate the efficiency, random-primed qPCR Human Reference cDNA (Takara Bio, Mountain View, CA, USA) was used. ACTB, GUSB, and CDKN1B were selected as endogenous controls using GeNorm software (https://genorm.cmgg.be/ accessed on 9 July 2015) for tissue analysis, whereas ACTB and GAPDH were selected as endogenous controls for exosome samples. Relative gene expression levels were expressed as the ratio of target gene expression to the geometric mean of the endogenous gene expressions, according to the Pfaffl formula [33 (link)].

RTqPCR was performed to analyze the relative expression of 51 CSC-related genes on a Roche LightCycler^®480 II system (Roche Ltd., Basel, Switzerland) (Supplementary Table S2). RNA from cell pellets and frozen tissue samples was extracted using standard TRIZOL (Invitrogen) method. Reverse transcription reactions were performed from 1.0 µg of total RNA using random hexanucleotides and a High-Capacity cDNA (complementary DNA) Reverse Transcription Kit (Applied Biosystems, USA) following the manufacturer’s instructions. The thermal cycling conditions were as follows: 10 min at 25 °C, 120 min at 37 °C, and 5 s at 85 °C. RTqPCR was performed with assays based on hydrolysis probes using 1 µL of cDNA, TaqMan Gene Expression Master Mix, and a TaqMan Gene Expression Assay (Applied Biosystems, USA) in a 5 µL final reaction volume. The thermal cycling parameters were as follows: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. For efficiency calculations, we used random-primed qPCR Human Reference cDNA (Clontech, USA). ACTB, GUSB, and CDKN1B were selected as endogenous controls using GeNorm software. Relative gene expression levels were expressed as the ratio of target gene expression to the geometric mean of the endogenous gene expressions according to Pfaffl formula^{23 (link)}.