Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress

Figures & data

Figure 1. Specificity of real-time PCR amplification. Melting curves (dissociation curves) of the 9 reference gene amplicons after the real-time PCR reactions, all showing one peak. X-axis (horizontal): temperature (°C); Y-axis (vertical): negative derivative of fluorescence over temperature (-dF/dT).

Table 1. Name and function of genes mentioned in the text.

Symbol	Gene name	Function
ACTB	Actin, beta	Major component of the non-muscle cytoskeleton
HMBS	Hydroxymethylbilane synthase	Heme synthesis porphyrin metabolism transferase activity
HPRT1	Hypoxanthine phosphoribosyl transferase 1	Purine synthesis in salvage pathway
RPL13	Ribosomal protein L13	Ribosomal protein that is a component of the 60S subunit
RPL32	Guanine nucleotide binding protein (G protein), ribosomal protein L32	Ribosomal protein that is a component of the 60S subunit
18SrRNA	18S ribosomal RNA	Biogenesis and export of the 40S ribosomal subunit
		Basal transcription machinery
TBP	TATA box binding protein	Coordinates initiation of transcription in core promoters
TFRC	Transferrin receptor (p90, CD71)	Encoding transferrin receptor protein 1 that is required for iron import from transferrin into cells
	Tyrosine 3-monooxygenase/tryptophan	Signal transduction
	YWHAZ	5-monooxygenase activation protein, zeta polypeptide

Table 2. Primers used for quantitative real-time PCR analysis of chicken mRNAs.

Target	Primers	PCR Product	Accession No.
ACTB	5′-AGCGAACGCCCCCAAAGTTCT-3′ 5′-AGCTGGGCTGTTGCCTTCACA-3′	139 bp	NM_205518.1
HMBS	5′-CATAGGTCAGCTCCTGAGCCC-3′ 5′-CCCATCCTGCAGGGCAGATAC-3′	121 bp	XM_417846.5
HPRT1	5′-CGCTCCATGGCGACTCACA-3′ 5′-GCCAGTCTCTCTGTCCTGTCC-3′	155 bp	NM_204848.1
RPL13	5′-GGCCCGTGTTATCTCAGAGG-3′ 5′-GGATCCCAAAGAGACGAGCG-3′	89 bp	NM_204999.1
RPL32	5′-GCAGGTCCTGTCAGCTTCGT-3′ 5′-GGCGTCTCGGGAGAATGGAT-3′	76 bp	NM_001252255.1
18SrRNA	5′-ATGGTGACCACGGGTAACGG-3′ 5′-GCTGCCTTCCTTGGATGTGG-3′	84 bp	HQ873432.1
TBP	5′-CCATTGCACTTCGTGCCC-3′ 5′-GCCAGTCTGGACTGTTCCTC-3′	154 bp	NM_205103.1
TFRC	5′-GGACCGCCTGTCATCTTCTGG-3′ 5′-GCCTCACCAGCCTCAAAGGAG-3′	122 bp	NM_205256.2
YWHAZ	5′-AGGAGCCGAGCTGTCCAATG-3′ 5′- CTCCAAGATGACCTACGGGCTC-3′	84 bp	NM_001031343.1

ACTB: β-actin; HMBS: hydroxymethylbilane synthase; HPRT1: hypoxanthine phosphoribosyl transferase 1; RPL13: ribosomal protein L13, RPL32: ribosomal protein L32, 18SrRNA: 18S ribosomal RNA; TBP: TATA box binding protein; TFRC: Transferrin receptor; YWHAZ: tyrosine 3-monooxygenase / tryptophan 5-monooxygenase activation protein zeta.

Table 3. Candidate reference genes ranking in the uterus of heat-stress and control hens.

Rank	geNorm (M value)	Best Keeper (correlation coefficient)	NormFinder (Stability value)	All programs (mean)^a
1	YWHAZ (0.494)	YWHAZ (0.967)	YWHAZ (0.049)	YWHAZ (0.192)
2	HPRT1 (0.494)	HPRT1 (0.956)	HPRT1 (0.060)	HPRT1 (0.199)
3	RPL13 (0.748)	HMBS (0.944)	HMBS (0.131)	HMBS (0.350)
4	HMBS (0.864)	TFRC (0.927)	RPL13 (0.148)	RPL13 (0.352)
5	TBP (0.933)	RPL13 (0.833)	TFRC (0.160)	TFRC (0.401)
6	TFRC (0.972)	ACTB (0.825)	TBP (0.192)	ACTB (0.554)
7	ACTB (1.102)	18SrRNA (0.733)	ACTB (0.385)	TBP (0.662)
8	RPL32 (1.402)	RPL32 (0.153)	RPL32 (0.548)	RPL32 (0.932)
9	18SrRNA (1.644)	TBP (0.139)	18srRNA (0.606)	18SrRNA (0.994)

ACTB: β-actin; HMBS: hydroxymethylbilane synthase; HPRT1: hypoxanthine phosphoribosyl transferase 1; RPL13: ribosomal protein L13, RPL32: ribosomal protein L32, 18SrRNA: 18S ribosomal RNA; TBP: TATA box binding protein; TFRC: Transferrin receptor; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta. ^aMean of each gene = [(M value)+(Stability value)+(1-correlation coefficient)]/3.

Table 4. Candidate reference genes ranking in the ovary of heat-stress and control hens.

Rank	geNorm (M value)	Best Keeper (correlation coefficient)	NormFinder (Stability value)	All programs (mean)^a
1	YWHAZ (0.787)	YWHAZ (0.965)	HPRT1 (0.130)	YWHAZ (0.317)
2	HPRT1 (0.806)	HPRT1 (0.963)	YWHAZ (0.133)	HPRT1 (0.324)
3	HMBS (0.818)	HMBS (0.957)	TFRC (0.133)	TFRC (0.354)
4	TFRC (0.846)	TBP (0.942)	RPL13 (0.177)	HMBS (0.363)
5	RPL13 (0.886)	ACTB (0.941)	HMBS (0.227)	RPL13 (0.375)
6	TBP (0.966)	RPL13 (0.938)	TBP (0.255)	TBP (0.426)
7	RPL32 (1.272)	TFRC (0.918)	ACTB (0.282)	RPL32 (0.597)
8	18SrRNA (1.604)	RPL32 (0.795)	RPL32 (0.314)	ACTB (0.723)
9	ACTB (1.829)	18SrRNA (0.122)	18SrRNA (0.470)	18SrRNA (0.752)

ACTB: β-actin; HMBS: hydroxymethylbilane synthase; HPRT1: hypoxanthine phosphoribosyl transferase 1; RPL13: ribosomal protein L13; RPL32: ribosomal protein L32, 18SrRNA: 18S ribosomal RNA; TBP: TATA box binding protein; TFRC: Transferrin receptor; YWHAZ: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta. ^aMean of each gene = [(M value)+(Stability value)+(1-correlation coefficient)]/3.

Figure 2. The overall result (mean) of the most stable reference genes in both ovary and uterus of heat-stress and control hens.