Optimization of RNA isolation from human ocular tissues and analysis of prostanoid receptor mRNA expression using RT-PCR.
OBJECTIVE
The isolation and analysis of human ocular RNA is problematic due to variables such as rapid degradation, tissue composition, and melanin contamination. The purpose of this work was to optimize an extraction protocol for the isolation of intact total RNA from a variety of diverse human ocular tissues and to employ RT-PCR to assess the expression of mRNA coding for all eight prostanoid receptors.


METHODS
Total RNA was extracted from human iris, ciliary body, choroid, and retina using an RNeasy(R) Midi Kit. Total RNA was extracted from human cornea, sclera, and optic nerve using Tri-Pure(R) Isolation Reagent. 1.0 microgram of total RNA was reverse transcribed into cDNA and subsequently amplified by PCR (35 cycles) using primers designed against each of the human prostanoid receptor cDNAs. PCR products were analyzed by gel electrophoresis and endonuclease digestion.


RESULTS
The total yield and quality of RNA derived from each tissue varied according to tissue composition and the isolation method employed. RT-PCR analysis revealed that each tissue expressed all prostanoid receptor mRNAs, however, 50 cycles of PCR was required to visualize FP receptor expression in scleral tissue. In all cases, prostanoid receptor mRNA expression was significantly lower than in human nonpregnant myometrium, which was used as the positive control.


CONCLUSIONS
The different cellular composition of each ocular tissue ultimately dictated the methodology to be employed for the isolation of total RNA. Thus, two extraction protocols were optimized for the isolation of intact high quality RNA from a variety of human ocular tissues. The identification of all prostanoid receptor mRNAs in a diverse set of human ocular tissues suggests potential mechanisms for prostanoid-based therapeutics aimed at IOP reduction and stimulates speculation as to additional physiological and or pathophysiological roles mediated by prostanoids.
Kyveris
Angela
A
School of Optometry, University of Waterloo, Waterloo, Ontario, Canada. msenchyn@sciborg.uwaterloo.ca
Maruscak
Erin
E
Senchyna
Michelle
M
D002829:Choroid / Q000737:chemistry / Q000378:metabolism; D002924:Ciliary Body / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005123:Eye / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007498:Iris / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000235:genetics* / Q000378:metabolism; D012160:Retina / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D012333:RNA, Messenger; D011982:Receptors, Prostaglandin; D012313:RNA