Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green

Table 3 Detailed conditions used for real-time PCR assays.

Gene name	[primer]^a (μM)	[Mg⁺⁺]^b (mM)	Annealing conditions^c (°C…s)	Product TM^d (°C)	Amplicon Length^e (bp)
βactin	0.3	3.5	58°C…4s	83.5	97
CypA	0.4	4	58°C…5s	85.2	127
GAPDH	0.4	3	58°C…4s	86.9	202
HPRT	0.4	4	60°C…5s	84.7	123
IL1a	0.4	4	65°C…5s	81.5	85
IL1b	0.4	3.5	58°C…5s	82.0	79
IL1RA	0.4	3	65°C…5s	80.7	117
IL1R1	0.4	3	64°C…5s	80.0	105
IL1R2	0.4	4	64°C…5s	84.5	116
IL6	0.4	3	65°C…5s	80.9	79
IL6R	0.4	5	60°C…5s	84.3	117
TNFa	0.4	3	58°C…4s	84.7	111
TNFR2	0.4	4	60°C…5s	84.9*	83
SOCS3	0.4	4	65°C…5s	84.7	99
C/EBPβ	0.4	4	59°C…5s	90.2	90.2
cFos	0.4	4	60°C…5s	84.6	84.6
cJun	0.4	4	62°C…5s	89.2	89.2
COX-2	0.4	4	60°C…4s	83.8	83.8
NOS-2	0.4	4	58°C…5s	83.8	83.8

Abbreviations: see Tables 1, 2.^a final concentration of oligonucleotide primers (μM), ^b final concentration of Mg²⁺ (mM), ^c Annealing temperature (°C) and duration (s), ^d melting temperature of specific PCR product, ^e Amplicon length in base pairs. *TNF-r2: a second melting peak close to 88°C can coexist depending on template. The two peaks correspond to a single amplification product as controlled using high-resolution gel electrophoresis and PCR with recombinant and genomic DNA as templates.

ISSN: 1471-2172