Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Table 2 RT-qPCR reference gene primer sequences and characteristics for all candidate reference genes

Predicted gene product	Primer sequence	GC (%)	Tm(°C)^a	Amplicon (bp)	Ex-ExSpanning^b	Efficiency (%)	R²
JmjC	F: CCAGTTATGATTTCGGCAATAATGG	40.0	54.5	139	No	96.8	0.991
	R: GGTGTCAGTTCATCGTCTTCAG	50.0	55.4	139	No	96.8	0.991
Dynein	F: CGAAGCCAGTAGTGGTATCAAGG	52.2	57.1	84	No	98.0	0.991
	R: CGAATCAGGTTGTTCTGGAGTCG	52.2	57.6	84	No	98.0	0.991
Histone H3	F: GAAGCCTACCTGGTGGGTCTC	61.9	59.3	151	No	101.4	0.999
	R: CGTCCGATCACCTTCCGTCTC	61.9	59.5	151	No	101.4	0.999
Cyclophilin	F: GTAGGACAAAGCCAGCACAACAGG	54.2	60.2	83	No	99.0	0.997
	R: GAATGAATCGGTGCTCGTAGGAGG	54.2	59.2	83	No	99.0	0.997
Cyclophilin Ex-Ex	F: CTGGGTTTCAAGAGCCAACGAC	54.5	58.5	105	Yes	98.3	0.997
	R: CATCAATGCCGACGGACTGAAT	50.0	57.7	105	Yes	98.3	0.997
EF-1á	F: GGACTCTCCATCAAGGGTATTGC	52.2	57.3	150	No	98.4	1.000
	R: GTATCCAGGCTTGAGGACACC	57.1	57.4	150	No	98.4	1.000
PGK	F: GATGCCGAGAAGAAGGGTGTG	57.1	58.0	69	No	98.7	0.996
	R: CGAAGGAAATGCTTGTGTTGCGAC	50.0	59.2	69	No	98.7	0.996
eIF-2	F: GTGATGCGTGCTTGATTGCTTG	50.0	57.6	78	No	99.6	0.997
	R: CCTTCATGTCGTGGCGAAGC	60.0	59.0	78	No	99.6	0.997
ATPase^c	F: GGTGGTGATATTGCTCCCTTG	52.4	55.7	164	No	98.4	0.996
	R: CGTTGATCTTCACTGATCTTTAGTCG	42.3	55.4	164	No	98.4	0.996
Ubiquitin	F: CCTTCGTCGGAACATCACTACC	54.5	57.3	126	No	94.1	0.997
	R: CGTCAAGGGTGATAGTCTTGC	52.4	55.5	126	No	94.1	0.997
GAPDH	F: GACAACTTCCACAAGGTCATCTCC	50.0	57.5	83	No	100.3	0.999
	R: CTGGTGTAGACAGCCAAGTCG	57.1	57.6	83	No	100.3	0.999
18s rRNA	F: GTTGCCCGCCACTCTTTACGATTG	54.2	60.6	81	No	98.0	0.998
	R: GTATCAGTGCCAAGCCTCTGC	57.1	58.3	81	No	98.0	0.998
β-tubulin Ex-Ex^d	F: CCAAATTCTGGCAGGTCATG	50.0	54.2	114	Yes	100.8	0.998
	R: CTTGTCCCTCGTTGAAGTACAC	50.0	55.4

^aThe annealing temperature for all standard curve analyses was performed at 60°C to demonstrate the efficiency of the primer sets under our assay conditions; the calculated Tm values are provided for reference.
^b'Ex-Ex spanning’ refers to primers that span an exon-exon junction; these primer sets did not yield a product using gDNA as a template.
^cThe PSN0332 contig sequence had an extra “t” in the reverse primer region as compared to the Pseudo-nitzschia multiseries genome sequence, yet the primer set demonstrated high efficiency.
^dβ-tubulin was not used in the studies presented as it was not printed on the microarray. It is a validated exon-spanning primer set that demonstrated good efficiency, and may have value as a reference gene for future studies.

ISSN: 1471-2199