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cDNA synthesis

SD Solmaz Dini
MZ Mansoureh Zakeri
SE Shiva Ebrahimpour
FD Fariba Dehghanian
AE Abolghasem Esmaeili
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500 ng treated RNA with DNase I, was used for cDNA synthesis using a cDNA synthesis kit (Takara, Japan). The reaction in a final volume of 10 μL containing 500 ng RNA, 2 μL 5 × prime script buffer, 0.5 μL RT enzyme, 0.5 μL oligo dT primer, 0.5 μL random 6mer was incubated at 37 °C for 15 min, and at 85 °C for 5 s.

Moreover, cDNA synthesis for microRNA was performed using BON-miR miRNA 1st-strand kit (Bonyakhteh, Tehran, Iran, Cat No # BON209001) through polyadenylation. Briefly, elongation of microRNAs was performed in a polyadenylation reaction, with a final volume of 20 μL at 37 °C for 30 min. Then the reaction to make cDNA was performed immediately after polyadenylation reaction and using the existing compounds in BON-miR miRNA 1st-Strand cDNA Synthesis Kit, for each sample of polyadenylated RNA.

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