Sybr qpcr mix

To quantify COX-2 mRNA expression, total RNA was isolated following the instructions provided with TRIzol reagent (Invitrogen, CA). The total RNA yield and purity were determined by measuring the absorbance at 260 nm and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). cDNA was then synthesized using a PrimeScript RT reagent kit with gDNA Eraser (Takara Clontech, Japan) according to the manufacturer’s instructions. Real-time PCR was performed on a C1000 Touch™ thermal cycler instrument (Bio-Rad, Philadelphia, PA) with SYBR reagent (Takara, Dalian, China) following the manufacturer’s instructions. Amplification was performed according to the reported protocol with some modifications [32]. A 25-µL mixture of 12.5 µL SYBR qPCR Mix (Takara, Dalian, China), 2 µL PCR primers [33] mix (10 µM), 2 µL diluted template cDNA, and 8.5 µL deionized distilled water was processed for RT-PCR. The relative fold changes in COX-2 expression were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The PCR primers used in this study are listed in Supplementary Table 1.

We selected 101 pairs of fresh tumor and adjacent tissues to measure POC1A mRNA expression and used TRIzol reagent for extracting total RNA, which was further transcribed to cDNA through reverse transcription. RT-qPCR was performed with SYBR qPCR mix (Takara Bio Inc) in a 7500 real-time PCR System (Thermo Fisher Scientific). GAPDH, whose primers were 5’-CTCGCTTCGGCAGCACA-3’ (forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse), was regarded as an endogenous reference for normalization. Other primer information is shown in Supplementary Table 1. Finally, we used the 2-ΔCt formula to represent the expression of the genes above.

Complementary DNA (cDNA) was synthesized with Reverse Transcriptase M-MLV (RNase H-) (TaKaRa, Dalian, China; Code No. D2639A) using the oligo dT. Real-time PCR technology was employed to determine the mRNA levels of LDLR, LXRα and ABCG1 on the Light Cycler instrument (Roche Diagnostics, Germany) using the SYBR Green method. Each PCR mixture (final volume of 20 μL) was composed of 10 μL of SYBR qPCR Mix (TaKaRa, Dalian, China), 0.4 μL of each gene-specific primer, and 1 μL cDNA in each reaction. The primers used for real-time RT-PCR were as follows: LDLR: forward 5′-AGGAGTGCAAGACCAACGAG-3′—and reverse 5′-TACGTACCTCATGGCGGTTG-3′; ABCG1: forward 5′-CCTGTCTGATGGCCGCTTTC-3′ and reverse 5′-TCCCTCGGGTACGGAGTAAG-3′; LXRα: forward 5′-GAGTCATCCGAGCCTACAGC-3′ A and reverse 5′-AAGAATCCCTTGCAGCCCTC-3′ AGβ-actin: forward 5′-ACCCGCGAGTACAACCTTC-3′ and reverse 5′-ATGCCGTGTTCAATGGGGTA-3′. The thermal cycling parameters were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 45 s, and 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s. The relative expression of target genes was calculated using the 2^−ΔΔCT method (the analysis was performed by the ABI Prism 7300 SDS Software).

Approximately 2 μg of total RNA from the four stages were reverse-transcribed by M-MLV reverse transcriptase (Takara) using oligo (dT) as the primer. The unigenes of interest were subjected to quantitative real-time PCR (qRT-PCR) analysis. The primers and accession numbers of these genes and the internal reference gene (18S ribosomal RNA) are listed in S1 Table. The amplifications were performed using 0.4 μl (10 μM) of specific primers, 10 μl of SYBR qPCR Mix (Takara), 0.4 μl Rox (Takara) and 2.0 μl cDNA in a final volume of 20 μl. The cycling parameters were 95°C for 5 min followed by 30 cycles of 95°C for 5 s, 60°C for 15 s and 72°C for 20 s. Three independent biological replicates were performed for each gene tested in real time PCR reactions. The relative gene expression was analyzed using the 2^-ΔΔct method. The expression of the 18S ribosomal RNA gene (unigene comp170_c0, 100% similarity) was stable in the four developmental stages based on the RNA-seq data; this gene was therefore used as the internal reference (S2 Table).

The total RNA extraction of tissues and cells was performed using RNA extraction kits (QIAGEN, Germany) according to the manufacturer’s protocol. RT-qPCR was performed using the Bio-Rad CFX96 Real-Time PCR operating instrument with SYBR qPCR Mix (Takara, Dalian, China). The lncRNA and mRNA were normalized to the levels of GAPDH, and the miRNA was normalized to the levels of U6. The primers used for real-time PCR are shown in Supplementary Table 2.