. 2018 Nov 23;9(92):36474–36491. doi: 10.18632/oncotarget.26356

Table 4. Primers used for the evaluation of miRNA expression and miRNA target validation.

Primer		Sequence
miR-specific RT		CAGGTCCAGTTTTTTTTTTTTTTTVN
RNU6B	Forward	GTCGTGAAGCGTTCCA
RNU6B	Reverse	CAGGTCCAGTTTTTTTTTTTTTTTAAA
RNU48	Forward	ACCGCAGCGCTCT
RNU48	Reverse	TCCAGTTTTTTTTTTTTTTTGGTCA
miR140-5p	Forward	CAGCAGTGGTTTTACCCTATG
miR140-5p	Reverse	GGTCCAGTTTTTTTTTTTTTTTCTAC
miR140-3p	Forward	GTACCACAGGGTAGAACCA
miR140-3p	Reverse	GTACCACAGGGTAGAACCA
ITGB3-miR-140-3P	Forward	AAACTAGCGGCCGCTGAGCCACTGCCCCCGGCTGTGGTTGT
ITGB3-miR-140-3P	Reverse	CTAGACAACCACAGCCGGGGGCAGTGGCTCAGCGGCCGCTAGTTT
ITGB3 mutated-miR-140-3P	Forward	AAACTAGCGGCCGCTGAGCCACTGCCCCCGGCTAAGTTGT
ITGB3 mutated-miR-140-3P	Reverse	CTAGACAACTTAGCCGGGGGCAGTGGCTCAGCGGCCGCTAGTTT

In the sequences of the miR-specific RT primer, V = A or G or C, N = A or G or C or T. RNU6B and RNU48 were used for the normalization of the miRNA expression using a Sybr Green qRT-PCR miRNA assay. The predicted ITGB3 3’UTR target regions complementary to miR-140-3p was designed based on the prediction by the online tool TargetScan Human 7.0 (http://www.targetscan.org). The overhangs created by oligonucleotide annealing were designed to be complementary to those sites recognized by the restriction enzymes PmeI (GTTT^AAAC) and XbaI (T^CTAGA). NotI internal site (GC^GGCCGC) was added for cloning digestion.