Ophthalmic genetics · 2012
Human RNA integrity after postmortem retinal tissue recovery.
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Montanini Luisa, Ferrari Stefano, Crafa Pellegrino, Ghirardini Stella, Ponzin Diego, Orsoni Jelka G, Mora Paolo
Аннотация
PURPOSE: To assess the parameters for postmortem retinal tissue recovery and processing that affect the quality of RNA extracted from the retina/retinal pigment epithelium (RPE) complex.
METHODS: RNA was extracted from retina/RPE samples. The RNA quality was determined based on qualitative/quantitative measurements made with a Bioanalyzer (Agilent) and on the expression of a long retinal gene (RPE65). After a pilot analysis on rats, ocular RNA was extracted from human donor eyeballs (group A) explanted according to conventional procedures for cornea transplantation. In a second experiment, another group of human donor eyeballs (group B) were processed in a much shorter time. The postmortem interval (T) comprised two periods: T1, the time between a donor's death and enucleation, and T2, the time between eyeball explantation and immersion of the excised retina/RPE sample in preservative solution (T = T1 + T2).
RESULTS: A short T2 was correlated with good quality of RNA extracted from the retina/RPE complex (p = 0.043) and successful expression of a tissue-specific gene (p = 0.007). No other parameter appeared to influence RNA quality.
CONCLUSIONS: The time between eyeball explantation and immersion of the retina/RPE sample in preservative solution was the chief parameter affecting the quality of RNA extracted from the retina/RPE complex.
METHODS: RNA was extracted from retina/RPE samples. The RNA quality was determined based on qualitative/quantitative measurements made with a Bioanalyzer (Agilent) and on the expression of a long retinal gene (RPE65). After a pilot analysis on rats, ocular RNA was extracted from human donor eyeballs (group A) explanted according to conventional procedures for cornea transplantation. In a second experiment, another group of human donor eyeballs (group B) were processed in a much shorter time. The postmortem interval (T) comprised two periods: T1, the time between a donor's death and enucleation, and T2, the time between eyeball explantation and immersion of the excised retina/RPE sample in preservative solution (T = T1 + T2).
RESULTS: A short T2 was correlated with good quality of RNA extracted from the retina/RPE complex (p = 0.043) and successful expression of a tissue-specific gene (p = 0.007). No other parameter appeared to influence RNA quality.
CONCLUSIONS: The time between eyeball explantation and immersion of the retina/RPE sample in preservative solution was the chief parameter affecting the quality of RNA extracted from the retina/RPE complex.