A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

Table 1 Primer and probe sequences for amplification and detection

Template	Primer/oligo	Sequence 5′-3′	Modification	Amplicon/probe
Plasmid pEGFP-N1	CMV-EGFP-frw	GGGGCGGAGCCTATGGAAAA	-	PCR product: (5′ biotinyl-)CMV-EGFP, 1136 bp
	Btn-CMV-EGFP-frw	GGGGCGGAGCCTATGGAAAA	5′ biotin
	CMV-EGFP-rev	TGTCGCCCTCGAACTTCACCTC	-
CMV-EGFP	EGFPfrw1	TGAGCAAGGGCGAGGAGCTGTT	-	PCR/qPCR product, 295 bp
	EGFPrev1	AAGATGGTGCGCTCCTGGACGT	-	PCR/qPCR product, 295 bp
	EGFPfrw2	GTGACCACCCTGACCTAC	-	qPCR product, 83 bp
	EGFPrev2	ATGGCGGACTTGAAGAAG	-	qPCR product, 83 bp
	EGFP-LNA1	CAGtGCtTCaGCcGCTA*	5′ FAM, 3′ BHQ1, LNA-oligo*	qPCR dual labeled probe
HeLaScribe ‘Positive Control DNA’**	HELA NUCLEAR FW	CTCATGTTTGACAGCTTATCGATCCGGGC	-	PCR product: (5′ biotinyl-)HS-DNA, 1182 bp
	Btn-HELA NUCLEAR FW	CTCATGTTTGACAGCTTATCGATCCGGGC	5′ biotin
	HELA NUCLEAR RV	ACAGGACGGGTGTGGTCGCCATGAT	-
HS-DNA	HNqPCRfrw1	GCCGGGCCTCTTGCGGGATAT	-	qPCR product, 132 bp
	HNqPCRrev1	CGGCCAAAGCGGTCGGACAGT	-	qPCR product, 132 bp
	HNqPCRfrw6	GTCCATTCCGACAGCATCGCCA	-	qPCR product, 144 bp
	HNqPCRrev6	GGCTCCAAGTAGCGAAGCGAGC	-	qPCR product, 144 bp
	HN_SONDE1	TGGCGTGCTGCTAGCGCTAT	5′ FAM, 3′ BHQ	qPCR dual labeled probe

*Locked Nucleic Acid (LNA) - bases are depicted as lower case.
**‘Positive Control DNA’ is provided with the Promega ‘HeLaScribe Nuclear Extract in vitro Transcription System’. Its DNA sequence is provided within the Additional file 1 “HeLaScribe Positive Control DNA Sequence”.

ISSN: 1471-2199