Table 3. The final list of improved extension primers in the high-quality primer selection system.

Gene	SNP Position	Direction	Length (bp)	Refractory	SNP	Primer Sequence	Tm (°C)	GC (%)
Forward Direction
IL-1α	-889	Forward			C	GAAATTCTTTAATAATAGTAACCAGGCAACAC	57	31
IL-1α	-889	Forward			T	AGAAATTCTTTAATAATAGTAACCAGGCAACAT	56	27
IL-1β	-511	Forward			T	CCTGCAATTGACAGAGAGCTCCT	57	52
IL-2	+166	Forward			T	CACAGCTACAACTGGAGCATTTACTT	56	42
IL-4	-590	Forward			C	CACCTAAACTTGGGAGAACATTGTC	56	44
IL-4	-590	Forward			T	AAACACCTAAACTTGGGAGAACATTGTT	56	36
IL-10	-592	Forward			C	ATCCTGTGACCCCGCCTGTC	58	65
IL-10	-592	Forward			A	ATCCTGTGACCCCGCCTGTA	56	60
TNF-α	-308	Forward			G	GCAATAGGTTTTGAGGGGCATGG	57	52
Reverse Direction
IL-1β	+3962	Reverse			C	TAAGCCTCGTTATCCCATGTGTCG	57	50
IL-1β	+3962	Reverse			T	TAAGCCTCGTTATCCCATGTGTCA	56	46
IL-2	-330	Reverse			G	ATACAAAAGTAACTCAGAAAATTTTCTTTGTCC	56	27
IL-2	-330	Reverse			T	GATACAAAAGTAACTCAGAAAATTTTCTTTGTCA	56	26
IL-4	-1098	Reverse			G	CTCAGCTAATTAGGAAAAAGAGCTACC	57	41
IL-4	-1098	Reverse			T	CCTCAGCTAATTAGGAAAAAGAGCTACA	57	39
IL-6	-174	Reverse			C	TGCAATGTGACGTCCTTTAGCATG	56	46
IL-10	-819	Reverse			C	GAGCAAACTGAGGCACAGAGATG	57	52
IL-10	-819	Reverse			T	TGAGCAAACTGAGGCACAGAGATA	56	46
TNF-α	-238	Reverse			G	CTCCCCATCCTCCCTGCTCC	60	70
TNF-α	-238	Reverse			A	CTCCCCATCCTCCCTGCTCT	58	65
IL-1R	+1970	Reverse			C	CGCAGTGGTCGAGTCTGCAG	58	65
IL-1R	+1970	Reverse			T	GCGCAGTGGTCGAGTCTGCAA	58	62
IL-4Rα	1902	Reverse			G	TCCACCGCATGTACAAACTCCC	57	55
IL-4Rα	1902	Reverse			A	CTCCACCGCATGTACAAACTCCT	57	52
Length Modification
IL-1β	-511	Forward	24		C	TCCTGCAATTGACAGAGAGCTCCC	59	54
IL-2	+166	Forward	23		G	AGCTACAACTGGAGCATTTACTG	53	43
IL-6	-174	Reverse	22		G	CAATGTGACGTCCTTTAGCATC	53	45
IL-10	-1082	Forward	24		A	ACACTACTAAGGCTTCTTTGGGAA	56	42
IL-12	-1188	Reverse	24		C	TGTTTCAATGAGCATTTAGCATCG	52	38
IL-12	-1188	Reverse	23		A	GTTTCAATGAGCATTTAGCATCT	50	35
TGF-β1	codon 25	Forward	25		G	GCTCACTGGTGCTGACGCCTGGCCG	68	72
TGF-β1	codon 25	Forward	25		C	GCTCACTGGTGCTGACGCCTGGCCC	68	72
TNF-α	-308	Forward	22		A	CAATAGGTTTTGAGGGGCATGA	53	45
Refractory Mdification
IFN-γ	5644	Reverse		21(m-3)	A	GTGTGTGTGTGTGTGTGTGAT	52	48
IL-1RA	+11100	Forward		21(m-3)	C	TTCATCCGCTCAGACAGCGGC	58	62
IL-1RA	+11100	Forward		21(m-1)	T	GCCTTCATCCGCTCAGACAGTG	59	59

IL: interleukin, IFN: interferon, TNF: tumor necrosis factor, TGF: transforming growth factor, R: receptor, RA: receptor antagonist

The SNP site in each primer is underlined.

Each extension primer used in this study has a unique “tag” sequence in the 5′ region

(An “anti-tag” that is a complementary “tag” immobilizes on the microspheres).