Sybr green detection rt pcr system

Total RNA was extracted using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer's instructions. qRT-PCR was performed using a SYBR green detection RT-PCR system (Takara, Japan). Actin was utilized for normalization. All samples were analyzed in triplicate. The 2^−ΔΔCt method was used to calculate the relative RNA expression. The probes are listed below:
Forward primer: ATTGCATTGCCAATTTGA
Reverse primer: GTCAAATAAAGTTTGGAAAAC.

Tissues and cells were harvested and lysed with TRIzol (Sigma 93,289) following the manufacturer’s protocol. After purification and reverse transcription, cDNA was harvested. Specific primers were used for gene amplification. qPCR was performed using the SYBR Green detection RT‑PCR system (Takara Bio, Inc.) with RT mix (cat. no., RR036B; Takara Bio, Inc.) and SYBR Green (cat.no., 740,703; Takara Bio, Inc.). The thermocycling conditions were as follows: initial denaturation at 95°C for 5 s; 40 cycles at 95°C for 5 s of denaturation, 95°C for 35 s and 60°C for 30 s for annealing and elongation and 60°C for 30 s for final extension; and GAPDH was used for normalization. RNA was examined by detecting the absorption of OD260/OD230, RNA with ratio: > 2 was retained.
The following key primers were used:
hsa_circ_0001879 (F) CCCTCCAAGTCCAGTAAGAAGT
hsa_circ_0001879 (R) AGATCCTAAGAGGTGCGAGTTTA
miR-6873-5p (F): CTTCTCTGTAAGGCAAAGT
miR-6873-5p (R): CTCTACAGCTATTCCGAAC
HDAC9 (F): AGTAGAGAGGCAT
HDAC9 (R): GGAGTGTCCTTTCGG
U6 (F): CTCGCTTCGCAGCACA
U6 (R): AACGCTTCACGAATTTGCG
GAPDH (F) GGAGCGAGATCCCTCCAAAAT
GAPDH (R) GGCTGTTGTCATACTTCTCATGG

Total RNA was isolated from tissues and cells using the Qiagen RNeasy Mini Kit in combination with on-column DNase treatment (Applied Biosystems, USA). A High Capacity RNA-to-cDNA Kit (Applied Biosystems) was used to synthesize the first strand of cDNA. Quantitative real-time PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) with gene-specific primers. According to the manufacturer's instructions, the total RNA was extracted with TRIzol reagent (USA, NY, USA). qRT-PCR was performed using the SYBR Green Detection RT-PCR System (TaKaRa, Japan) using the following OLA1 primers: forward primer, ACTTTTTCACTGCAGGCCCA; reverse primer, GTACTTTCCAGCAGCCTTGAC. 18s rRNA was used as the reference control and was amplified with the following primers: forward primer, GTAACCCGTTGA ACCCCATT; reverse primer, CCATCCAATCGGTA GTAGCG. The relative mRNA expression level was determined by the 2^-ΔΔCt method. All qRT-PCR experiments were conducted in triplicate.

We extracted total RNA from HCC and ANLT tissues, and HCC cell lines using TRIzol (Invitrogen, NY, USA) according to the manufacturer’s instructions. QRT-PCR was performed using the SYBR green detection RT-PCR system (Takara, Japan) according to manufacturer’s instructions. The following primers were obtained from Servicebio Technology (Wuhan, China): AC092171.4 forward: 5’-ATTACCCCGCCCTGGATTTG-3’, reverse: 5’-TTGTTTTCCCCACCCC-3’; miR-1271 forward: 5’-CAGCACTTGGCACCTAGCA-3’, reverse: 5’-TATGGTTGTTCTCCTCTCTGTCTC-3’; GRB2 forward: 5’-GGGCCTTTCTTATCCGAGA-3’, reverse: 5’-TGCACATCGTTTCCAAACTT-3’; β-actin forward: 5’-CACCCAGCACAATGAAGATCAAGAT-3’, reverse: 5’-CCAGTTTTTAAATCCTGAGTCAAGC-3’; U6 forward: 5’-CTCGCTTCGGCAGCACA-3’, reverse: 5’-AAACGCTTCACGAATTTGCGT-3’. β-actin and U6 were used as internal controls. All samples were analyzed in triplicate. The relative mRNA, lncRNA, and miRNA expressions were determined using the 2^-ΔΔCt method. (Supplementary Figure 1)