Sybr primescriptmirna rt pcr kit

Paraffin sections 10 mm thick were cut, dewaxed, rehydrated and lightly stained with hematoxylin. Tumor tissues were examined and microdissected under a dissecting microscope (Leica ASLMD, Witts Baden, Germany). miRNA extraction was performed with a miRNeasy FFPE kit (Qiagen, Hilden, Germany), which enabled copurification of total RNA, including miRNA, from formalin-fixed and paraffin-embedded tissue sections. The primer sequences of human miR-432 and small nuclear RNA U6 were synthesized by GeneCopoeia, Inc. and its expression was measured using SYBR PrimescriptmiRNA RT-PCR kit and SYBR^® Green I (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. The universal small nuclear RNA U6 was used as an endogenous control for miRNAs. Total RNA extraction from cultured cells and the quantification of E2F3 and AXL was performed as described before [30 ]. Melting analysis of the PCR products was conducted to validate the amplification of the specific product. The primers of target genes were as followings: E2F3 (F): 5′-CAGGCTGGTTTCGGAAATGC-3′, E2F3 (R): 5′-TGGA CTTCGTAGTGCAGCTC-3′; AXL (F): 5′-CGCACTTACAAGACTTGGTCC-3′, E2F3 (R): 5′-GCTTCGCAGGAGAAAGAGGA-3′. GAPDH was used as internal loading control and its primer sequences were: GAPDH(F): 5′-GCACCGTCAAGGC TGAGAAC-3′, GAPDH (R): 5′-TGGTGAAGACGCCAGTGGA-3′.

Paraffin sections 10 mm thick were cut, dewaxed, rehydrated and lightly stained with hematoxylin. Tumor tissues were examined and microdissected under a dissecting microscope (Leica ASLMD, Witts Baden, Germany). miRNA extraction was performed with a miRNeasy FFPE kit (Qiagen, Hilden, Germany), which enabled copurification of total RNA, including miRNA, from formalin-fixed paraffin-embedded tissue sections. Expression of miR-573 was measured using SYBR Primescript miRNA RT-PCR kit and SYBR^® Green I (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. Melting analysis of the PCR products was conducted to validate the amplification of the specific product. The universal small nuclear RNA U6 was used as an endogenous control for miRNAs.