Rt2 ht first strand cdna synthesis kit

Leaf discs (0.78cm²) frozen and stored at −80 °C were lysed using the Qiagen TissueLyser II. RNA was extracted using the Trizol extraction method and in the presence of RNase inhibitor (Ambion). DNA was removed using the TURBO DNA free kit (Ambion), and RNA quantity and quality were determined using a NanoDrop (Thermo Scientific).
RNA (200ng) was reverse transcribed into cDNA using Qiagen’s RT² HT First Strand cDNA synthesis kit. RT–qPCR and melt curve analysis were performed on a Viia7 Real-time PCR system using the Power SYBR green PCR Master Mix (Thermo Fisher) according to the manufacturer’s instructions. Primers (Supplementary Table S1) were designed using Primer3 in Geneious R7.1.6, ensuring products spanned an intron. Primer amplification efficiencies were determined by the Ct slope method; efficiencies for all primer pairs were comparable (~95%) and no amplification was detected in the no template control. Relative fold change was calculated by the ΔΔCt method, using the average of three nulls as reference, as described by Livak and Schmittgen (2001) (link). The geometric mean of the Ct values for three reference genes was used for normalization (Vandesompele et al., 2002 ). Statistics were performed with SigmaPlot (version 11.0).

HeLa cells (1 × 106/well) were placed within 6 well plates and subjected to a medium containing IC50 concentrations of FE (3.25 mg/L) and LE (1.75 mg/L) for 24 h. After incubation, the isolation of total RNA was performed using a TriPure isolation reagent (Roche, USA). cDNA was synthesized with an RT2 HT First Strand cDNA synthesis kit (Qiagen, Germany). An RT2 SYBR Green qPCR Green I Master Kit (Qiagen) was used for quantitative analysis of gene expression. The
GAPDHgene was used to normalize the expression level of apoptosis-related genes
CASP3,
CASP8, and
CASP9. The primers used were 5ʹ -TGGAATTGATGCGTGATGTT-3ʹ and 5ʹ - TGGCTCAGAAGCACACAAAC-3ʹ for
CASP3, 5ʹ -TCCAAATGCAAACTGGATGA-3ʹ and 5ʹ -TCCCAGGA TGACC CTCTTCT-3ʹ for
CASP8, 5ʹ -CCA
TATGATCGA GGACAT CCA-3ʹ and 5ʹ -GACTCCCTC
GAGTCTCCA GAT-3ʹ for
CASP9, and 5ʹ -AGCCACATC
GCTCAGACAC-3ʹ and 5ʹ -GCCCAATACGACCAAATC
C-3ʹ for
GAPDH.