The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

BMC Research Notes

Table 1 Sequences of siRNAs, RNA oligos and RT-qPCR primers used in the study

*siRNAs*
Name	Passenger strand	Guide strand
RRM1-2	CCCAGUUACUGAAUAAGCAGAUCUU	AAGAUCUGCUUAUUCAGUAACUGGGCU
RRM1-3^§	GCAAACUCACUAGUAUGCACUUCUA	UAGAAGUGCAUACUAGUGAGUUUGCCU
ApoB-1^§	GUCAUCACACUGAAUACCAAU	AUUGGUAUUCAGUGUGAUGAmCmAC
ApoB1 mm control	GUGAUCAGACUCAAUACGAAU	AUUCGUAUUGAGUCUGAUCAmCmAC
81-control	AAGAUCUGCUUAUUCAGUAACUGGG	CCCAGUUACUGAAUAAGCAGAUCUU
*Single-strand RNA oligos*
Name	Sequence
RRM1-3-sense	GCAAACUCACUAGUAUGCACUUCUA
RRM1-3-antisense	UAGAAGUGCAUACUAGUGAGUUUGCCU
*qPCR Primers*
Target	Forward	Reverse
RRM1(A)^†	TGGATCAAGGTGGGAACAAG	CGACGAGAAGGAAAGGACAC
RRM1(B)^†	GGTACAAGGTCTGGCAGATGCT	TTCCAGTGTCGACCGAAGGT
RRM1(C)^†	CATCCACATTGCTGAGCCTAAC	GGGTCAGAAGTTTGGGACGAA
ApoB1 site	AGCCATGGGCAACTTTACCT	AAAGGAAATGGGCAACGATA
ApoB external	GGCACTGTGGGTCTGGAT	TTCTTCTCTGGAGGGGACTG
HMBS	AGCCTGTTTACCAAGGAGCT	GAGTGAACAACCAGGTCCAC
LMNA	TGAGGCCAAGAAGCAACTTCA	CTCATGACGGCGCTTGGT
Polr2a	TTACTCCCCTGCATGGTCTC	TGGGAGACATAGCACCACCT

* Represents phosphorothioate linkages between nucleotides in the siRNA backbone. ^† Targets RRM1-A, B and C represent the cleavage sites for the various RRM1-specific siRNAs, as shown schematically in Figure 1A. ^§RRM1-3 and ApoB1 are complementary in sequence to both human and mouse target genes. Accession numbers: RRM1, NM_001033.3; LMNA, NM_005572.3; HMBS, NM_000190.3; ApoB, NM_009693.2; Polr2a, NM_009089.2. Amplicon sizes: RRM1(A): 114 bp; RRM1(B): 127 bp; RRM1(C): 79 bp; LMNA: 139 bp; HMBS: 65 bp; ApoB1 site 107 bp; ApoB external: 86 bp; Polr2a: 95 bp

ISSN: 1756-0500