Cdna synthesis kit

Total RNA was extracted from THP-1 cell line in each tested well using total RNA extraction kit (Yekta Tajhiz Azma, Tehran, Iran) in accordance with the manufacturer’s protocol [37 (link)]. Extracted RNA was purified by DNase (Thermo Fisher Scientific) treatment, and its concentration was calculated by NanoDrop (NanoDrop Technologies, USA). After adjustment complementary DNA (cDNA) was synthesized using cDNA synthesis kit (Yekta Tajhiz Azma, Tehran, Iran).
To analyze the expression of levels of atg5, atg7, atg12, lc3b, and β-actin (BACT) genes, amplification of corresponded genes was performed using Rotor-Gene Q (Qiagen, Germany) thermocycler. The reaction mixture of 20 µl contained 1 µL of each cDNA sample, 10 µl SYBR green qPCR master mix 2X (Ampliqon, Denmark), and 0.5 µL of each primer [37 (link)]. The final volume was adjusted by adding RNase/DNase-free water. The thermal cycling conditions consisted of an initial denaturation of 10 min at 95℃ followed by 40 cycles of 95℃ for 20 s, 59–61 °C for 30 s, 72℃ for 20 s and a final extension step at 72℃ for 20 Sect. [37 (link)]. The melt curve analysis was performed for each gene to rule out nonspecific amplifications. Relative expression level of each gene was compared to the β-actin gene, and results were analyzed using the 2^{− ΔΔCt} method incorporated into the relative expression software (REST).

The extraction process of total RNA was done from cell samples using RNX-PlusTM (Cinnagen, Tehran, Iran). The concentration of RNA was evaluated by a Nanodrop spectrophotometer (Varian, Australia) at 260/280 nm wavelength. Then, cDNA was synthesized by the cDNA Synthesis kit (Yekta Tajhiz Azma, Tehran, Iran). Using qRT-PCR, the expression level of anti-apoptotic and pro-apoptotic genes was determined in DU-145 transfected and non-transfected cells by SYBR Premix Ex Taq™ kit (Takara Bio, Japan). The characteristics of the genes used are demonstrated in Table 1. GAPDH gene was selected as endogenous control. The cycling conditions inclusive: initial denaturation at 95 °C for 5 min, 30 cycles for denaturation at 94 °C for 40 s, annealing at 64 °C for 40 s, and extension at 72 °C for 40 s, followed by 72 °C for 5 min for duplication of unfinished fragments. Analysis of gene expression was performed using (2^-ΔΔCt) method.

B16F10 cell line was cultured in DMEM medium containing 10% v/v FBS and 1% w/v penicillin–streptomycin. After sufficient proliferation, cells were trypsinized and prepared for injection into female C57BL6 mice (n = 5). A total of 5 × 10⁵ cells in 60 μl PBS were injected subcutaneously into the right side of each mouse. Fourteen days after injection, mice were sacrified by cervical dislocation. Tumor tissues were examined to assess the expression of IDO using conventional PCR technique. RNA was extracted from the target tissues using an RNA isolation kit (DENA ZIST ASIA) and the concentration of RNA was measured using nanodrop at 260 and 280 nm wavelengths. The extracted RNAs were converted to cDNA using cDNA Synthesis kit (YektaTajhizAzma). IDO specific primers were designed and synthesized by the German company Metabion. We used Taq DNA polymerase enzyme. Thermal cycling conditions used were: 95 °C for 5 min followed by 40 cycles at 60 °C for 1 min, 72 °C for 30 s and 45 °C for 30 s with total run time of approximately 2 h. Qualitative expression of the PCR product (123 bp) was assessed by 2% agarose gel electrophoresis.

The osteogenic gene expression was evaluated after 21 and 28 days. After washing microcapsules by the PBS, samples were fractured gently. Then, Trizol reagent was used for the extraction of total RNA according to the manufacture instruction. The gel electrophoresis and Nanodrop (Thermo Scientific, Waltham, MA, USA) were applied for determining the yield and value of the extracted RNA. 1 μl of the total synthesized RNA was used for the cDNA synthesis by CDNA synthesis kit (YektaTajhizAzma, Iran). Synthesized cDNA, syber green master mix, and designed primers were mixed based on the manufacture instruction. Primer sequences, melting temperature, and amplicon size of the designed primers were offered in Table 1. Cells were cultured on the polystyrene culture surface of the T75 flasks for the control group. All the experiments were repeated three times.
Table 1

Sequences, melting temperature, and amplicon size of primers used for RT-PCR

Target	Sense and antisense sequences5′ to 3’	tA (°C)	bp
BMP-2	F: GAGAAGGAGAGGCAAAGAAAG	61/59	183
	R:GAAGCAGCAACGCTAGAGAC
Osteocalcin	F: ATTGTGGCTCACCCTCCATCA	60	119
	R: AGGGCTATTTGGGCGTCATC
Osteonectin	F:GCAGAGGAAACCCAAGAGGAG	60	208
	R: TGGCAAAGAAGTGGCAGCAG
RUNX-2	F:ACCTTGACCATAACCGTCTTC	67/57	145
	R: GGCGGTCAGAGAACAAACTA
DSPP	F:CTGGTGCATGAAGGTCATAGAG	57	90
	R: CAATTTGCGGATCTCAGAGG
GAPDH	F: GAAGGTGAAGGTCGGAGTC	65	100
	R: GAAGATGGTGATGGGATTTC