Protecting RNA in fixed tissue: an alternative method for LCM users.
RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.
Kihara
Alexandre Hiroaki
AH
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900 São Paulo, SP, Brazil. kihara@icb.usp.br
Moriscot
Anselmo Sigari
AS
Ferreira
Paula Juliana
PJ
Hamassaki
Dânia Emi
DE
D000818:Animals; D005404:Fixatives / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry; D015870:Gene Expression / Q000502:physiology; D007834:Lasers; D051379:Mice; D042282:Microdissection / Q000379:methods*; D011108:Polymers / Q000737:chemistry; D012313:RNA / Q000032:analysis* / Q000737:chemistry; D012160:Retina / Q000737:chemistry / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; D012313:RNA; C003043:paraform