Isolation of total-RNA from formalin-fixed rat retina.
The aim of this project was to establish a method for the purification of total-RNA from fixed rat-retina. Two different established methods were used for RNA purification, and successful isolation was verified with RT-PCR for amplification of beta-actin (two different product-lengths) and subsequent gel-electrophoresis. Total-RNA was successfully isolated from fixed rat-retina. The house keeping gene, beta-actin could be detected after fixing the retina either with 1% formalin or with 4% paraformaldehyde (PFA). Hexamer-primer based RT-PCR gave better results than the oligo-d(T)-primer based RT-PCR method. Both the 698 and 225 bp beta-actin-fragments could be successfully amplified, where amplification of the latter was more efficient. This approach shows that tissue fixation prior to RNA-isolation facilitates the rapid isolation of undamaged RNAs in tissues such as the retina, which are known to yield low levels of RNA and are vulnerable to RNases.
D000818:Animals; D005260:Female; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000737:chemistry / Q000187:drug effects*
D005404:Fixatives; D005557:Formaldehyde; D012313:RNA