Gen elute mammalian genomic total rna kit

Total RNA isolation was carried out by using Gen Elute Mammalian Genomic Total RNA Kit (Sigma) from untreated and quercetin-treated HeLa cells (25 and 50 µM for 48 h) and cDNA was synthesized (ABI RT-PCR Kit). The synthesized cDNA was then used as a template for TaqMan® Human Apoptosis Array (Thermo Fisher), which has a range of different apoptosis regulators from both intrinsic and extrinsic pathways. A TaqMan-based custom array was designed consisting of several tumor suppressor genes and regulatory genes from various signal transduction pathways. PCR array was run on QuantStudio3 and analyzed by the ΔΔC_T method using DataAssist™ software from Thermo Fisher. The data were normalized using 18s rRNA expression (apoptosis array) and global normalization (custom array). RQ indicates the fold change in gene expression against untreated control after normalization with the selected endogenous gene.

A total of 2x10⁶ cells were plated and treated with 50 µM genistein for 48 h. Gen Elute Mammalian Genomic Total RNA Kit (Sigma, St Louis, MO, USA) was used to obtain total RNA from genistein treated and untreated HeLa cells. RT-PCR Kit (ABI, Waltham, MO, USA) was used to synthesize cDNA which was subsequently used for the array. TaqMan-based array was customized with primers specific for several genes involved in signal transduction pathways as well as TSGs. PCR array was run on QuantStudio3 and analyzed by the Comparative Delta Delta Ct method (ΔΔCT method) using DataAssist^TM software v3.01 (ThermoFisher, Waltham, MO, USA) with global normalization. RQ signifies the relative fold change in gene expression of treated sample with respect to untreated control. The statistical significance was calculated as per the mean of three experiments using two-tailed t-test with p ≤ 0.05.

RNA was isolated from the treated (20, 30 and 50 µM for 48 h) and control cells as per the kit’s protocol (Gen Elute Mammalian Genomic Total RNA Kit; Sigma, USA). The qualitative check was performed by running the isolated RNA in 1% agarose gel and the same was quantitated using nanodrop (Nanodrop 2000c; Thermo Scientific™, Waltham, MA, USA). The RNA was used to synthesize cDNA with the help of a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA USA) as per the manufacturer’s instruction. TaqMan^® Gene Expression Array and master mix (Apoptosis Array, cat. No. 4414072 and 4369514) was employed to evaluate the expression of various molecular targets involved in apoptosis and signalling pathways, including cell proliferation, cell survival, etc. To each well of the assay plate, 10 µL of cDNA (100 ng/well) from the treated cells, along with 10 µL of the master mix, was added. The plate was then subjected to run for qPCR (QuantiStudio3; Applied Biosystems) and analysis was performed by DataAssistTM software version 3.01 (ThermoFisher Scientific) with the 2^−ΔΔCq method. The expression level of GAPDH (a housekeeping gene) was used to normalize the data. The RQ values display the fold change for the expression of various genes in treated cells compared with the untreated control.