Superscript 4 reverse transcriptase kit

Total RNA was extracted from PC tissues and cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). mRNA was reverse-transcribed into cDNA using the SuperScript IV Reverse Transcriptase kit (Thermo Fisher Scientific, Inc.), while miRNA was reverse transcribed into cDNA using TaqMan™ MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). qPCR was subsequently performed using a PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Inc.). All the kits were used according to the manufacturer's instructions. GAPDH was used as the reference gene for IGF-1 expression, while U6 was used as the reference gene for miR-7515 expression. The following primer sequences were used for the qPCR: miR-7515, 5′-AGAAGGGAAGATGGTGAC-3′; IGF-1 forward, 5′-GCTCTTCAGTTCGTGTGTGGA-3′ and reverse, 5′-GCCTCCTTAGATCACAGCTCC-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′; U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′. The reaction conditions were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 1 sec and 60°C for 60 sec. Relative expression levels of the target mRNA or miRNA were determined using the 2^−ΔΔCq method (19 (link)).

The nucleotide sequences of designed primers for the target and normalizer genes are shown in Table 1. Total RNA was extracted and purified using the Qiagen RNeasy Mini Kit (Cat. No: ID: 74104; Qiagen, Germany) according to the manufacturer's protocol. Quality and concentration assessments of the RNA samples were carried out using a Thermo Scientific NanoDrop One (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Thermo Scientific SuperScript IV Reverse Transcriptase kit was used for cDNA synthesis. We chose the ABL gene as a normalizer gene, and relative expression of the Ang1, Ang2, Ang4, Tie1, and Tie2 genes were measured in BM samples of ALL patients and in normal samples. A total reaction volume of 15 mL was used for quantitative real-time polymerase chain reaction (qPCR). We used 7.5 mL of RealQ Plus Master Mix Green, without Rox (AMPLICON, Odense, Denmark) in each reaction, which was performed in the ABI StepOne Plus system in duplicates. As the first step of the qPCR, initial activation was performed at 95°C for 10 min. The thermal cycling program was carried out for 40 cycles with the following: denaturation (95°C for 10 s), annealing (58–64°C depending on the gene for 20 s), and extension (72°C for 30 s). After cycling, the final extension step (72°C for 10 min) was performed.