Protecting RNA in fixed tissue: An alternative method for LCM users

https://doi.org/10.1016/j.jneumeth.2005.04.019Get rights and content

Abstract

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.

Introduction

Analysis of gene expression is a valuable tool for the assessment of normal and pathological tissue condition (Medhurst et al., 2000, Bernard and Wittwer, 2002, Fortina et al., 2002, Gutala and Reddy, 2004, Torres et al., 2004). Laser capture microdissection (LCM) of stained frozen sections has been used successfully to obtain pure cell populations in order to determine specific gene expression patterns in tissues with a complex mixture of cell types (Emmert-Buck et al., 1996, Bonner et al., 1997, Luo et al., 1999, Suarez-Quian et al., 1999, Burbach et al., 2003, Volgin et al., 2004). However, RNA degradation is a major drawback in most common fixation protocols recommended for tissue processing prior to LCM (Goldsworthy et al., 1999, Craven et al., 2002, Qin et al., 2003). Degradation is usually caused by the nature of chemical components utilized and the time consumed in several fixation steps, resulting in poor quality and/or limited total RNA yield (Foss et al., 1994, Abrahamsen et al., 2003). Several RNA isolation kits especially developed for LCM samples are commercially available, but usually are expensive.

The main purpose of this study was to test the reliability of an alternative fixation and RNA isolation protocol for analyzing gene expression in LCM samples. Since ideal fixation must provide acceptable morphology (Ginsberg and Che, 2004, Mojsilovic-Petrovic et al., 2004), allow proper laser capture of selected cells (Goldsworthy et al., 1999) and maintain the integrity of RNA (Craven et al., 2002), the proposed protocol was evaluated in these terms.

Section snippets

Tissue collection and fixation

Adult mice were sacrificed with an overdose of ketamine (30 mg/100 g of body weight, i.m., Parke-Davis, Ann Arbor, MI, USA) and xylazine (2 mg/100 g, i.m., West Haven, CT, USA). Experiments were conducted in accordance to the Guide for the Care and Use of Laboratory Animals and the Biomedical Sciences Institute/USP guidelines. Subsequently, eyes were dissected out and retinas were submitted to different protocols (for details, see Table 1). Retinas were incubated (Groups 2 and 4) or were not

Effects on RNA yield and quality

Spectrophotometer analysis indicated that incubation in RNAlater™ prior to fixation increased RNA yield in about four-fold (P < 0.01, Fig. 1A). Additionally, improvement in RNA quality was also found (P < 0.01, Fig. 1B). The use of oyster glycogen followed by freezer incubation also increased RNA yield (P < 0.01, Fig. 1A), but did not bring extra benefit in RNA quality (Fig. 1B). However, RNAlater™ treatment combined to the use of oyster glycogen followed by freezer incubation resulted in a strong

Discussion

One important aim in understanding the molecular basis of normal or pathological tissue condition is the quantification of gene expression (Medhurst et al., 2000, Bernard and Wittwer, 2002, Gutala and Reddy, 2004). LCM of stained frozen sections has been used successfully to obtain pure cell populations in tissues formed by a complex blend of cell types (Emmert-Buck et al., 1996, Bonner et al., 1997, Luo et al., 1999, Suarez-Quian et al., 1999, Burbach et al., 2003). In order to identify

Acknowledgements

This research was supported by FAPESP, CNPq and PRONEX/MCT. A.H.K. was the recipient of fellowship from FAPESP. The authors would like to thank Marley Januário da Silva, Pryscila de Rossi, Antonio G. Soares Jr., Sidney Veríssimo Filho, Leandro M. de Castro for the excellent technical assistance and Marinilce F. Santos and Chao Yun Irene Yan (from Institute of Biomedical Sciences, University of Sao Paulo) for their helpful comments.

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