The HepG2 cells were cultured in six-well plates at a density of 1.5 × 105. After 24 h of adhesion, the culture media were aspirated off and the cells were treated with the nanospheres. After 24 h of incubation, the media were removed and the treated cells were washed with ice-cold PBS. Fastlane Cell cDNA kit (QIAGEN, Germany) was used to prepare cDNA directly from the cultured cells, according to the manufacturer’s instructions. The mRNA levels of tp53, CAS-3, -9 FADD and BCl-2 as well as the reference gene, GAPDH, were assayed using gene-specific SYBR Green-based QuantiTect Primer assays (QIAGEN, Germany). Real-time PCR reactions and analyses were performed on in Applied Biosystems 7500 Fast (Foster City, CA, USA). The PCR reaction conditions, reaction volumes and calculation of fold change in expression were chosen to be exactly the same as published earlier (Hasan et al., 2011 (link)).
Sybr green based quantitect primer assays
Manufactured by Qiagen
Sourced in Germany
SYBR Green-based QuantiTect Primer assays are a set of pre-designed primer pairs for performing quantitative real-time PCR (qRT-PCR) experiments. They are designed to specifically target and amplify regions within genes of interest. The assays utilize the SYBR Green I dye, which binds to double-stranded DNA, allowing for the quantification of gene expression levels.
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3 protocols using sybr green based quantitect primer assays
1
Apoptosis Gene Expression in HepG2 Cells
2
Gene Expression Quantification Protocol
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For quantification of gene expression after stimulation, up to 1 μg RNA was used for cDNA synthesis using QuantiTect® Reverse Transcription Kit (Qiagen®, Hilden, Germany) according to the manufacturer's instructions. For qPCR analysis, a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems™, Foster City, USA) was used. qPCR was performed using either a TaqMan®-based assay (Actb: Mm00607939_s1; Tnfα: Mm00443258_m1; Mki67: Mm01278617; LgR5: Mm00438890_m1; Smoc2: Mm00491553_m1; Clca4b: Mm01616360_m1; Applied Biosystems®, Foster City, USA) or SYBR™ Green-Based QuantiTect® Primer Assays (Actb: Mm_Actb_2_SG; Cldn4: Mm_Cldn4_1_SG; Cldn8: Mm_Cldn8_1_SG; Ocln: Mm_Ocln_1_SG; Tjp1: Mm_Tjp1_1_SG; Cxcl1: Mx_Cxcl1_1_SG; Cxcl2: Mx_Cxcl2_1_SG; Cxcl5: Mm_Cxcl5_2_SG; Qiagen®, Hilden, Germany). Each sample was measured in triplicate. Actb was used as endogenous reference control gene. Relative gene expression was calculated using the 2−ΔCt method.
3
Quantitative Analysis of Gene Expression
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The qPCR-based quantification of the gene expression levels of the cDNA samples was performed using a TaqMan®-based singleplex assay with Actb (Mm00607939_s1) as the endogenous control gene, Mki67 (Mm01278617) and Slc5a1 (Mm00451210_m1) as the target, and the following TaqMan®-based multiplex assays: 4-plex 1 [Actb (Mm00607939_s1_qsy_ABY) as the endogenous control gene, Cldn2 (Mm00516703_s1_VIC; data not shown), Cldn7 (Mm00516817_m1_qsy_JUN; data not shown), and Tnfα (Mm00443258_m1_FAM)] and 4-plex 2 [Cldn4 (Mm_00515514_s1_qsy_ABY), Cldn8 (Mm00516972_s1_qsy_JUN; data not shown), Ocln (Mm00500912_m1_FAM), and Tjp1 (Mm01320638_m1_VIC)] (all from Thermo Fisher Scientific, Waltham, MA, USA). SYBR® Green-based QuantiTect Primer Assays (Qiagen®, Hilden, Germany) were used for Actb (Mm_Actb_1_SG) as the endogenous control gene, and Chga1 (Mm_Chga_1_SG) and Muc2 (Mm_Muc2_2_SG) as target genes. Each sample was either measured in duplicate or triplicate using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA). Relative quantification (RQ) was performed using the 2−ΔΔCT method [16 (link)].
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