Molecular Vision: Tea, Mol Vis 2013; 19:501-508. Table 1

Table 1. Small ncRNA suitability as reference genes for normalization of miRNA expression in rat retinal samples.

Small ncRNA	Species reactivity	Assay ID	Amplification efficiency in rat retina	Order of magnitude	R² value	Mean Ct
4.5S RNA(H)* “Variant 1”	Rat	001716	49.3%	2.4	1.00	24.1
4.5S RNA(H)* “Variant 5”	Rat	001717	ND – low abundance	ND	ND	ND
snoRNA202*	Mouse	001232	ND – low abundance	ND	ND	ND
snoRNA*	Rat	001718	ND – low abundance	ND	ND	ND
U6 snRNA*	Human, mouse and rat	001973	56.2%	4.2	1.00	20.9
RNU6B*	Human	001093	ND – low abundance	ND	ND	ND
Y1*	Rat	001727	ND – low abundance	ND	ND	ND
U87*	Rat	001712	62.0%	2.4	0.98	23.8
5S rRNA	Rat	N/A	92.5%	4.0	0.98	23.0
hsa-miR-16 ⁺	Human, mouse and rat	000391	77.5%	4.2	0.98	24.2
mmu-miR-379⁺	Human, mouse and rat	001138	ND – low abundance	ND	ND	ND
hsa-miR-191⁺	Human, mouse and rat	002299	ND – low abundance	ND	ND	ND
hsa-let-7d⁺	Human, mouse and rat	002283	ND – low abundance	ND	ND	ND

Thirteen small ncRNAs were tested for miRNA normalization. Applied Biosystems assay ID numbers are provided for all small ncRNAs other than 5S rRNA, which was not available as an Applied Biosystems TaqMan assay. PCR primer amplification efficiencies were determined using serial dilutions of the standard RNA pool to a minimum of 2 orders of magnitude to generate a standard curve. The order of magnitude of the standard curve dilution series, the R² value for each standard curve and the mean Ct across all test samples, including the standard pool is shown. N/A=not available; ND=not able to be determined; *=small ncRNA assays referred to as endogenous controls by Applied Biosystems; +=miRNA identified from in-house Exiqon microarrays as being stably expressed with strain and oxygen exposure.