Sybr green quantitative rt qpcr kit

Total RNA was extracted using Trizol (Thermo Fisher Scientific) according to the instruction. Then reverse transcription was performed with superScript III Reverse Transcriptase (Thermo Fisher Scientific) and PCR reaction systems were prepared with SYBR Green Quantitative RT-qPCR Kit (Roche) according to the instructions. All primers used in this study were purchased from Sangon and as follows:
HAND2-AS1 Forward: Forward: 5′-GGAGTCACAGGCAGTCGTAGA-3′
HAND2-AS1 Reverse: 5′-GAAGGCACAGATCATTCATGG-3′
β-actin Forward: 5′-TTCCAGCCTTCCTTCCTGGG-3′
β-actin Reverse: 5′-TTGCGCTCAGGAGGAGCAAT-3′

To confirm the effect of BAPTA on apoptosis related gene expression, 40 μM of BAPTA were applied for 30 minutes after bead rolling. After 12 hours, damaged neurons were collected for the qPCR. GAPDH gene was used as an internal standard. To extract the RNA, trizol reagent (Thermo Fisher) was used. Total RNA was reverse‐transcribed into complementary DNA (cDNA) using the cDNA synthesis kit (Thermo Fisher) following the manufacture's protocol. PCR reaction was carried out using the SYBR® Green Quantitative RT‐qPCR Kit (Roche, Switzerland) and a Roche real‐time PCR system (LightCycler® 96). ΔCt values were calculated by subtracting the GAPDH Ct value from that of target genes. Relative expression levels were calculated using the 2^−ΔΔCt method. Primer sequences and the size of amplicons are listed in Table 1.