Impairment of IGF2 gene expression in prostate cancer is triggered by epigenetic dysregulation of IGF2 -DMR0 and its interaction with KLF4

Table 2 Primer sets used for pyrosequencing, qMSP, RT-qPCR, ChIP-qPCR and for LOI and CNV studies

Assay	Primer identity	Primer sequence (5′-3′)	PCR-size (bp)
Pyro-sequencing	IGF2-DMR0-PS-F	TTTTTTGTTGTATTTTGGATTTAGATTTTTT	186
	IGF2-DMR0-PS-R	CTCCAAACACCCCCACCTTAA
	IGF2-DMR0-Seq	GTGGGGAGGGGGTTTATTTTT
	IGF2-H19-ICR-PS-F	GGGGGCTCTTGCATAGCACATGGGTA	242
	IGF2-H19-ICR-PS-R	ATAAACCCTTATCCTATAAATACCCTCA
	IGF2-H19-ICR-PS-Seq	GGTATTTTTGGAGGTTTTTTT
qMSP	GSTP1-MSP-U-F	GATGTTTGGGGTGTAGTGGTTGTT	91
	GSTP1-MSP-U-R	CCACCCCAATACTAAATCACAACA	91
	GSTP1-MSP-M-F	TTCGGGGTGTAGCGGTCGTC	97
	GSTP1-MSP-M-R	GCCCCAATACTAAATCACGACG	97
RT-qPCR	IGF2-RTPCR-F	GGGCAAGTTCTTCCAATATGA	214
	IGF2-RTPCR-R	TCACTTCCGATTGCTGGC	214
	ßActin-RTPCR-F	CGGAGTACTTGCGCTCAGGAGGA	226
	ßActin-RTPCR-R	CCTTCCTTCCTGGGAATGGAGTC	226
	KLF4-RTPCR-F	ACTCGCCTTGCTGATTGTCT	127
	KLF4-RTPCR-R	AATTGGCCGAGATCCTTCTT	127
	AMACR-RTPCR-F	ACGACTTACAGGACAGCAGA	181
	AMACR-RTPCR-R	CCTTCGTCTTCTCTGCAAAT	181
ChIP-qPCR	IGF2-DMR0-ChIP-F	CACCCTGGGGCCAAGGCAGT	100
ChIP-qPCR	IGF2-DMR0-ChIP-R	CTTGAGGGGTCATGGCACGGAAT	100
LOI study	IGF2-LOI-ApaI-F	CACCCCCCTCTTTCTCTTCT	245
LOI study	IGF2-LOI-ApaI-R	TACTGGGTCCCTCTGACTGCT	245
CNV study	ZNF80-GCN-F	CTGTGACCTGCAGCTCATCCT	120
	ZNF80-GCN-R	TAAGTTCTCTGACGTTGACTGATGTG	120
	GPR15-GCN-F	GGTCCCTGGTGGCCTTAATT	101
	GPR15-GCN-R	TTGCTGGTAATGGGCACACA	101

Abbreviations: qMSP quantitative methylation specific PCR, RT-qPCR reverse transcription of RNA in cDNA followed by quantitative real-time PCR, ChIP-qPCR chromatin immunoprecipitation followed by quantitative real-time PCR, IGF2-DMR0 differential methylated region in promoter P0 of the insulin-like growth factor 2 gene, IGF2/H19-ICR imprinting control region of IGF2 and H19 genes, KLF4 Kruppel-like factor 4, AMACR Alpha-methylacyl-CoA racemase, LOI loss of imprinting, CNV copy number variation, bp-base pairs