Sybr greener qpcr supermix universal kit

Real-time PCR was carried out to determine β-arrestin1 and β-arrestin2 gene expression in tumour and nontumour liver tissue samples and HCC cell lines with stepwise metastatic potential. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), and complementary DNA (cDNA) was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania), according to the manufacturer’s instructions. Real-time PCR was carried out using a Real-time PCR Detection System (ABI 7500) using an SYBR GreenER qPCR SuperMix Universal Kit (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. GAPDH cDNA amplification was used as an internal control for all real-time PCR amplification reactions. The primer sequences for each gene were as follows: β-arrestin1, forward primer: 5′-GGTAATAGATCTCCTTATCC-3′ and reverse primer: 5′-CCACAAGCGGAATTCTGTG-3′; β-arrestin2, forward primer: 5′-CCACGTCACCAACAATTCTG-3′ and reverse primer: 5′-TTGGTGTCTTCGTGCTTGAG-3′; and GAPDH, forward primer: 5′-TCAAGAAGGTGGTGAAGCAG-3′ and reverse primer: 5′-AGGTGGAAGAATGGGAGTTG-3′. The cycle threshold value was defined as the PCR cycle number at which the reporter fluorescence crossed the threshold. The cycle threshold value of each product was determined and normalized against that of the internal control, GAPDH.

To determine the ATF5 gene expression after the treatment of ATF5-CaP-rHDL, a
qPCR study was performed. For the analysis, C6 cells (5 ×
10⁴ per well) were seeded in 6-well plates (Corning, Corning,
NY, USA) and allowed to grow for 12 h, and then treated with different
formulations at the concentrations of 100 nM siRNA at 37 °C
for 48 h. Total cell RNA was extracted with an RNeasy Mini Kit (Qiagen,
Valencia, CA, USA), and cDNAs were synthesized with a SuperScript II reverse
transcriptase assay (Invitrogen, Carlsbad, CA, USA). qPCR was performed with a
SYBR GreenER qPCR SuperMix Universal kit (Invitrogen). Reactions were run with a
standard cycling programme: 50 °C for 2 min, 95 °C
for 10 min, 40 cycles of 95 °C for 15 s, and
60 °C for 1 min, on a Lightcycler 480II real-time PCR system
(Roche Diagnostics, Foster City, CA, USA).

Total RNA was extracted from sample triplicates and subjected to cDNA synthesis using random hexamers (Invitrogen). SYBR^® GreenER™ qPCR SuperMix Universal Kit (Invitrogen) was used for qPCR, and reactions were conducted using a Mx3005 P Real-Time PCR System (Agilent Technologies, CA, USA). To amplify the common CDS and specific 5′ UTRs of the two dsx1 transcripts, previously described primers²⁴ were used. To amplify the mCherry CDS, the mCherry_qPCR_F and mCherry_qPCR_R pair (Supplementary Table S1) was used. To amplify the housekeeping L32 gene, the DmagRPL32-realtime-5 and DmagRPL32-realtime-3 pair (Supplementary Table S1) was used.

Total RNA extraction from cell lines or frozen tissues was performed by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) for mRNA analysis. The miRcute miRNA First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) was used for miRNA analysis. Real-time PCR assay for mRNA analysis was performed by using the SYBR Green ER qPCR Super Mix Universal Kit (Invitrogen), and miRNA analysis was performed by using the miRcute miRNA qPCR Detection Kit (Tiangen, Beijing, China) in the StepOnePlus Real-Time PCR System (Applied Biosystems). Primers used for real-time PCR are listed in Table S2. The primer for miR-497 was purchased from Tiangen (Beijing, China). U6 and GAPDH were used as controls for the detection of miRNA and mRNA, respectively.

Real time PCR analysis was performed according to the supplier protocol (Invitrogen, California, USA) using Superscript III First-Strand Synthesis Supermix for qRT-PCR and SYBR GreenER qPCR Supermix Universal kit in 20 μL volumes per well of 96-well clear optical reaction plates. The components of reaction were SYBR Green PCR Master Mix (Invitrogen, California, USA), cDNA template, forward and reverse primers (Table 1), and nuclease-free water (Sigma, USA). PCR reactions were performed in Light Cycler 480 Real Time PCR instrument and analyzed according to accompanying software instructions (Roche Diagnostics Ltd., Switzerland). Beta-actin was used as an internal control and used to normalize ratios between samples. For primer pair, melting curve analysis was performed according to the instrument software instructions. Program was an initial incubation of 50°C for 2 min hold (UDG incubation) and 95°C for 10 min followed by 40 cycles at 95°C for 15 s, 60°C for 60 s. Relative change in mRNA level between control and treated groups were calculated by using 2^−ΔΔC_T method.