Table 4. Gene name, primer name, accession number, primer sequence, amplicon length, used primer concentration, annealing temperature and encountered efficiency of each candidate reference gene and the 3′:5′ assay target sequence used in the present study.

Gene name	Primer name	Accession nr.	Primer sequence	Amplicon length	Primer conc. (μM)	Ta (°C)	Efficiency (%)
actb	actb	AJ537421.1	F: CTGGGATGACATGGAGAAGA R: CTTGATGTCACGCACGATTT	406	0.32	57	100
actb	actb′	AJ537421.1	F: GTGCGTGACATCAAGGAGAA R: GCTGGAAGGTGGACAGAGAG	436	0.04	57	88
faua	faua	FM004681	F: GACACCCAAGGTTGACAAGCAG R: GGCATTGAAGCACTTAGGAGTTG	149	0.03	57	101
rpl13a	rpl13a	DT044539	F: TCTGGAGGACTGTCAGGGGCATGC R: AGACGCACAATCTTGAGAGCAG	148	0.08	57	100
tubb2	tubb2	FM003484	F: GCCTCAGGTGGCAAATATGT R: CCTCAGTGTAGTGACCCTTG	166	0.32	57	106
ywhab	ywhab	AM973424	F: TCTGCCAGGACGTGCTGAACC R: TCCACCGTATCCTTCTTTGAGTC	151	0.16	57	97
ef1a	ef1a	FM019753	F: AAATGCGGAGGAATCGACAA R: GAGCCCTTGCCCATCTCAG	70	0.32	57	102
rps18	rps18	AM490061	F: AGGGTGTCGGCAGACGTTAC R: CTTCTGCCTGTTGAGGAACC	163	0.16	57	98
gapdh	gapdh	AY863148	F: GTGCCAGCCAGAACATCAT R: TGTCGTCATATTTGGCGGGTTTC	171	0.32	57	100
actb	actb5′	AJ537421.1	F: CTGAACTACCCCATCGAGCA R: GACTTGGGGTTTCGGTTGTC	149	0.2	60	100
actb	actb3′	AJ537421.1	F: AAGATCATTGCCCCACCTGA R: CCTGCTTGCTGATCCACATC	100	0.2	60	99

Note that the actb primers used for the normalization (act and actb′) are different from the actb primers used for the 3′:5′ assay (actb5′ and actb3′).