Light cycler real time pcr detection system

For mRNA detection, total RNA was isolated from the serum samples, brain tissues, or harvested cells by using TRIzol total RNA isolation kit (Tiangen, Beijing, China). Quantitative RT-PCR was performed using the One-Step SYBR^® PrimeScript™ RT-PCR kit (Takara, Dalian, China) on the Lightcycler Real-time PCR detection system (BioRad, Hercules, CA). GAPDH was used as an internal control. Primer sequences were as follows: 5′- ATGAGGAATCTGGCTGCTGT-3′ (SLUG, forward), 5′- CAGGAGAAAATGCCTTTGGA-3′ (SLUG, reverse), 5′- TCGGAGTCAACGGATTTGGT-3′ (GAPDH, forward), and 5′- TTGGAGGGATCTCGCTCCT-3′ (GAPDH, reverse). The TaqMan Human miRNA Assays were applied for miR-203 detection. Briefly, 5 ng of total RNA was reversely transcribed to cDNA with stem-loop primers by using the TaqMan miRNA Reverse Transcription Kit (Ambion, Carlsbad, California, USA). Quantitative real-time PCR (qRT-PCR) was carried out by TaqMan Universal PCR Master Mix. All PCR primers were from the TaqMan miRNA Assays. U6 RNA was used as an internal control.

Total RNA was isolated from 100 mg of leaf tissue using a Total RNA Isolation System (Promega, Madison, WI, USA). The first-strand cDNAs were synthesized using the GoScript Reverse Transcription System (Promega). The gene expression level was quantified on a light cycler real-time PCR detection system (Bio-Rad) with SYBR Premix Ex TaqTM (TaKaRa, Kyoto, Japan). The sequences of gene-specific primers used for the qRT-PCR are presented in Table S1. The qRT-PCR reactions were performed in duplicates for the three biological replications of each treatment. The relative expression level of target genes was calculated from threshold values (Ct), using actin as the internal control.

Total RNA was isolated from 200 mg leaf tissue using the SV Total RNA Isolation System (Promega). First-strand cDNAs were synthesized using the GoScript Reverse Transcription System (Promega). The gene expression level was quantified on a light cycler real-time PCR detection system (Bio-Rad) with SYBR Premix Ex TaqTM (TaKaRa, DALIAN). The sequences of primers are presented in Supplementary Table S1. All the quantifications were normalized to ACTIN. The qRT-PCR reactions were performed in triplicates for each of the three independent samples. Quantification of the relative transcript level was performed using the 2^-ΔΔC_t method (Livak and Schmittgen, 2001 (link)).