Assessing Gene Expression in Lung Subcompartments Utilizing In Situ RNA Preservation

Gregory L. Baker*,1, Michael A. Shultz{dagger},{ddagger}, Michelle V. Fanucchi*, Dexter M. Morin{ddagger}, Alan R. Buckpitt{ddagger} and Charles G. Plopper*

* Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616; {dagger} Global Research, Amersham Biosciences, Sunnyvale, California 94086; and {ddagger} Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616

Received May 16, 2003; accepted September 22, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms of toxicant-mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity, significant differences and similarities in gene expression are observed throughout lung subcompartments. RNA-based technologies such as real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant-induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor-intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high-quality RNA could be preserved in situ over a period of time to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Two hours after 1-nitronaphthalene treatment, rat lungs were inflated with and stored in RNA preservation solution and stored at 4°C for 7 days. RNA was isolated from the lung subcompartments isolated by microdissection. After 7 days of storage, the RNA was intact, of high quality, and could be used for real-time RT-PCR to examine heterogeneous gene expression in the lung subcompartments. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high-quality RNA that may be used with molecular RNA-based technologies that will significantly accelerate our understanding of pulmonary injury and repair mechanisms.

Key Words: lung; heterogeneity; microdissection; RNA; preservation; real-time PCR; airways; parenchyma.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Current understanding of the mechanisms of toxicant-mediated pulmonary injury and subsequent repair have been aided through the use of molecular techniques, such as real-time polymerase chain reaction (PCR) and cDNA microarray technologies, which allow the quantification of gene expression. These technologies are critically dependent on the ability to repeatedly obtain intact, high-quality RNA from tissues.

Standard techniques of RNA isolation require that the tissues be processed immediately following necropsy or quick-frozen in liquid nitrogen until RNA isolations can be performed. These requirements limit the number of animals that can be necropsied and processed in a single day. Frequently, when investigators are designing experiments they are faced with balancing the need to obtain RNA from lung subcompartments and collecting tissues for other types of analysis. This is particularly problematic for inhalation exposure studies, where many animals are exposed and need to be necropsied immediately. Time constraints and limited laboratory personnel dictate that gene expression is frequently measured in whole-lung tissues rather than site-specific lung subcompartments. Unfortunately, gene expression analysis performed in this manner can skew the data measured toward representing other subcompartments, diluting and masking focal site-specific areas of injury and the changes in gene expression associated with the injury.

Previously a technique was reported for the successful isolation of high-quality RNA from site-specific lung subcompartments (Royce et al., 1996Go). However, this approach required that microdissections and RNA isolations be performed immediately, with additional steps added to the RNA extraction protocol. Experiments designed to examine changes in gene expression in response to pulmonary toxicants had to utilize staggered treatment schedules because only small numbers of animals could be processed for RNA isolation in a single day.

The present study presents a simplified technique for preserving RNA in situ such that subcompartment microdissections can be conducted later by using RNAlater to inflate and store lungs. RNA synthesis and degradation are stopped immediately and subcompartment RNA is stabilized in situ, providing investigators with a precise snapshot in time of lung subcompartment gene expression. With appropriate storage, the lungs remain patent, and microdissections may be delayed after collecting lung tissues without RNA degradation. We found that with RNAlater the isolated lung subcompartment RNA is intact, of high quality, and may be used with real-time reverse transcription PCR (RT-PCR) for examining lung subcompartment gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
All experiments were performed under animal protocols approved by the IACUC committee of the University of California, Davis. Adult male Sprague-Dawley rats were euthanized with an overdose of pentobarbital sodium (120 mg/kg, Sigma, St Louis, MO). A midline abdominal incision was made and the inferior vena cava was cut to exsanguinate the rat. Next, the trachea was exposed, a cannula was sutured into place just below the pharynx, and the trachea and lungs were removed from the body. The lungs were inflated with 15–20 ml of RNAlater (Ambion, Austin, TX) and stored immersed in RNAlater at 4°C until site-specific lung subcompartment sampling was performed.

1-nitronaphthalene treatment.
Additional male Sprague–Dawley rats were given a single intraperitoneal injection of 1-nitronaphthalene (1-NN, Aldrich, St Louis, MO) (100 mg/kg) dissolved in a corn-oil carrier (10 ml/kg). Two hours following injection the rats were euthanized, and their lungs were removed and processed as described above.

Lung subcompartment sampling procedure.
Site-specific lung subcompartment microdissections were performed 7 days following inflation of the lungs with RNAlater. The left lobe was separated from the remaining lobes, and the surface was blotted dry and glued to a glass coverslip with cyanoacrylate glue (Nexaband, Veterinary Product Laboratories, Phoenix, AZ). The coverslip-mounted lobe was placed in a petri dish, and a small piece of modeling clay was used to hold the coverslip in place while site-specific lung subcompartment microdissections were performed with a dissecting stereomicroscope. The site-specific subcompartments were isolated as illustrated in Fig. 1Go. The lateral edge of the left lobe, which does not contain airways, was cut away and used as the parenchymal compartment. Blunt dissection of the remaining parenchymal tissue was used to expose the airways.



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FIG. 1. Diagrammatic representation of microdissected lung subcompartments from the left lobe of the rat lung. The parenchymal compartment was collected from the outer lateral edge that does not contain airway subcompartments.

 
RNA isolation.
Total RNA was isolated using the RNeasy kit with on-column DNase digestion according to the manufacturer’s instructions (Qiagen, Valencia, CA). RNA was quantified spectrophotometrically by measuring OD260, and the purity was accessed by measuring OD260/OD280 ratios and electrophoresing RNA samples on 1% denaturing agarose gels. The gels were stained with ethidium bromide and imaged on a Typhoon 8600 Fluorescence Scanner (Amersham Biosciences Corp., Piscataway, NJ). The gel images were analyzed with Scion Image (Scion Corporation, Frederick, MD) using the Gelplot2 macro supplied with the program to measure the 28S to 18S ribosomal RNA bands ratio (28S/18S ratio).

Real-time RT-PCR.
All RT-PCR reagents were purchased from Applied Biosystems (Foster City, CA). RNA (200 ng) was reversed transcribed in a 25 µl reaction mixture containing 1X TaqMan RT Buffer, 5.5 mM MgCl2, 500 µl dNTPs, 2.5 µl random hexamers, 0.4 U/µl ribonuclease inhibitor, and 1.25 U/µl multiscribe reverse transcriptase. The reaction mixtures were incubated at 25°C for 10 min, 37°C for 60 min, and 95°C for 5 min. Primer and probe sequences were designed using Primer Express software (Applied Biosystems, Foster City, CA) and are listed in Table 1Go. The specificity of the primer probe sets was determined by agarose gel electrophoresis of PCR reaction products revealing a single band of expected size. Individual PCR reactions contained 1X TaqMan Mastermix, 1.25 µl of cDNA, 400-nM 5' and 3' primers, and 100 nM probe. PCR reactions were performed with an ABI 5700 sequence detection system using the following cycling protocol: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C 1 for min. The results were calculated using the comparative Ct method as described previously (Applied Biosystems, 2001Go). Briefly, the threshold cycle, Ct, is defined as the point at which the first significant increase in fluorescence is observed. The {Delta}Ct value is defined for each sample as the difference between the test gene and the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPD), (Cttest gene - CtGAPD). The {Delta}{Delta}Ct values of individual samples were then normalized to a calibrator sample for each gene by subtracting the {Delta}Ct(experimental sample) from the {Delta}Ct(calibrator sample). The reference calibrator sample used in these studies was whole-lung tissue RNA. Results are expressed as a fold difference in gene expression relative to whole-lung tissue RNA and were calculated using the formula: fold change = 2-{Delta}Ct.


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TABLE 1 Primers/Probes Used for Real-Time PCR Analysis
 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Site-Specific Sampling
The lungs remained inflated and the airways were patent immediately following inflation of the lungs with RNAlater. The lungs had a rubber-like texture. The parenchyma could be easily peeled away from the airways when site-specific lung subcompartments were microdissected from the left lung lobe. The RNA isolated from the lung subcompartments were of high quality, as their OD260/OD280 ratios averaged greater than 1.8, agarose gel electrophoresis of the RNA from these subcompartments showed that the samples were physically intact, and the 28S/18S ribosomal RNA ratios ranged from 1.8 to 2.2 (Fig. 2Go). From each site-specific lung subcompartment a minimum of 1 µg of total RNA (range 1.2 µg in terminal airways to 21 µg in parenchyma) was repeatedly isolated.



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FIG. 2. Representative formaldehyde gel electrophoresis of RNA isolated from fresh and preserved lung subcompartments. Denaturing agarose gel (1%, stained with ethidium bromide) of 500-ng of total RNA isolated from fresh whole lung and microdissected lung subcompartments stored at 4°C for 7 days in RNAlater. Lanes 1, fresh whole lung; 2, stored whole lung; 3, stored trachea; 4, stored proximal airway; 5, stored midlevel airway; 6, stored distal airway; and 7, stored parenchyma.

 
Normal Lung Gene Expression
The genes chosen for real-time RT-PCR analysis were selected because their expression patterns are known to be heterogeneous in lung subcompartments. Gene expression of cytochrome P450 2F4 (CYP2F4) has not been reported in the rat, but the mouse homologue (CYP2F2) expression is greatest in the airways in the mouse (Shultz et al., 2001Go). Calcitonin gene related peptide (CGRP) gene is predominately expressed in airway pulmonary neuroendocrine cells (Cadieux et al., 1986Go). The catalytic and regulatory subunits of gamma-glutamylcysteine synthetase ({gamma}GCS) are involved in the de novo synthesis of the cellular antioxidant glutathione, and their gene expression has not been reported in lung subcompartment in the rat, but {gamma}GCS enzyme activity has been reported in the mouse and was found at similar levels throughout the lung subcompartments (West et al., 2000Go). Real-time PCR analysis of RNA obtained from the microdissected airways showed that the trachea and intrapulmonary airways contained 18–150-fold more CYP2F4 gene expression than the whole lung and was barely detectable in lung parenchyma (Fig. 3AGo). CGRP gene expression was detected in the whole-lung samples. However, the trachea and intrapulmonary airways had two to six-fold more CGRP expression than the whole-lung RNA samples (Fig. 3BGo). CGRP was not detected in the lung parenchyma. Whole-lung tissue contained both CYP2F4 and CGRP gene expression, but in comparison to isolated airway subcompartments it is clear that the quantification of both genes was partially masked by RNA extracted from other subcompartments of the whole lung. Unlike CYP2F4 and CGRP, the catalytic and regulatory subunits of {gamma}GCS were expressed at similar levels throughout all lung subcompartments (Figs. 3CGo and 3DGo).



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FIG. 3. RNA measurements obtained from normal whole lung and lung subcompartments. Relative expression of CYP2F4 (A), CGRP (B), {gamma}GCS catalytic subunit (C), and {gamma}GCS regulatory subunit (D) in lung subcompartments. Note the heterogeneity in gene expression in the lung subcompartments. All measurements were normalized to whole-lung RNA.

 
Lung Subcompartment Gene Expression following 1-NN Treatment
Two hours after 1-NN treatment, the rats were dyspneic. Real-time PCR analysis of the rat lungs and their subcompartments revealed considerable differences in gene expression. The same genes were examined as in the normal untreated rat lungs. In addition, heme oxygenase 1 (HO1) and palate, lung, and nasal epithelium clone (PLUNC) were also examined. HO1 has recently been identified from microarray studies of the entire intrapulmonary airway tree as having the greatest increase in expression in the airways 2 h following 1-NN treatment (Shultz et al., 2003Go). PLUNC has been identified as being predominately expressed in the proximal portion of the tracheobronchial airway tree and is thought to play a role in host defense (Bingle and Craven, 2002Go).

Following 1-NN treatment, CYP2F4 was increased in the trachea four-fold and decreased two- to three-fold within the proximal and midlevel airways (Fig. 4AGo). CGRP was decreased five-fold in the trachea, increased in the airways two to four-fold, and was not detectable in the parenchyma (Fig. 4BGo). The catalytic and regulatory subunits of {gamma}GCS were increased 3–10-fold in whole lung, trachea, and intrapulmonary lung subcompartments compared to the control (Fig. 4C and DGo). HO1 expression increased in the whole lung, trachea and lung subcompartments 7–50-fold (Fig. 4EGo). PLUNC was found mainly in the trachea and was increased approximately five-fold in 1-NN treated rat trachea (Fig. 4FGo).



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FIG. 4. RNA measurements obtained following 1-NN treatment in whole lung and subcompartments. Relative expression of CYP2F4 (A), CGRP (B), {gamma}GCS catalytic subunit (C), {gamma}GCS regulatory subunit (D), heme oxygenase 1 (E), and PLUNC (F) in lung subcompartments. Note the heterogeneity in gene expression in the lung subcompartments. All measurements were normalized to carrier-treated control whole-lung RNA. Light gray bars = corn oil treated control rats; dark gray bars = 1-NN treated rats.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The conducting airways of the lung are a series of branching tubes that begin at the trachea and terminate at the terminal bronchioles. The airway tree demonstrates considerable spatial heterogeneity by airway position in cellular composition (Plopper et al., 1989Go), susceptibility to injury (Duan et al., 1993Go; Plopper et al., 1994Go, 2001Go), and the ability to repair following injury (Lawson et al., 2002Go; Van Winkle et al., 2001Go). The premise under which this study was undertaken is that, for defining the molecular processes underlying focal cellular events, it is extremely important to reproducibly sample the same lung subcompartments to detect focal site-specific changes in gene expression. Precision microdissection allows specific regions of the lung subcompartments, including their surrounding microenvironment, to be collected and studied.

Real-time RT-PCR analysis of CYP2F4, CGRP, and the catalytic and regulatory subunits of {gamma}GCS in normal lung demonstrated that gene expression can be either drastically different or highly similar among subcompartments in the lung. The increased levels of CYP2F4 and CGRP gene expression in the trachea and proximal airway segments are in agreement with observations made in the mouse (Shultz et al., 2001Go). The site-specific differences in gene expression of {gamma}GCS catalytic and regulatory subunits by subcompartment has not been reported in the rat or any other species, but our results are in agreement with the similarities in {gamma}GCS enzyme activity previously reported in lung subcompartments of the mouse (West et al., 2000Go).

The injury caused by the bioactivated pulmonary toxicant 1 NN has been described in Paige et al.(1997)Go and Sauer et al.(1997)Go. The injury is airway site-specific (Paige et al., 2000Go) and characterized by both ciliated and nonciliated Clara cell damage as well as an inflammatory cell infiltrate (Sauer et al., 1997Go). The most severe injury occurs in the trachea, and the midlevel airways have the lowest threshold for injury (Paige et al., 2000Go). The metabolism of 1 NN involves both P450-mediated bioactivation and glutathione metabolism (Halladay et al., 1999Go). The present study revealed heterogeneous gene expression by lung subcompartment 2 h following 1-NN treatment whether the same gene was examined in multiple compartments or multiple genes were analyzed within the same compartment. CYP2F4 gene expression had the greatest increase in the trachea while it decreased or remained unchanged in the intrapulmonary compartments. The large amount of CYP2F4 present in the trachea following 1-NN treatment may partially account for the epithelial damage and denudation previously reported (Paige et al., 1997Go), as mouse CYP2F2 can metabolize 1 NN in the mouse (Shultz et al., 2001Go). Whether the same is true in the rat has yet to be determined. {gamma}GCS catalytic and regulatory subunits were increased following 1-NN treatment in whole lung, trachea, proximal midlevel and distal airways while the parenchyma had no change in the catalytic subunit and the regulatory subunit was decreased. Proliferation and increased staining of CGRP-positive neuroendocrine cells has been previously reported following acute lung injury (Stevens et al., 1997Go). The present study similarly found that CGRP expression increased in the whole lung and intrapulmonary subcompartments and decreased five-fold in the trachea following 1-NN treatment. HO1 was increased in 1-NN treated rat whole lungs and different lung subcompartments, but not to the same degree, and has been described as being protective in a recent review (Otterbein et al., 2003Go) via its ability to break down heme into CO, Fe++, and biliverdin. PLUNC is expressed mainly in the nose, trachea, and proximal intrapulmonary airways (Weston et al., 1999Go). Following 1-NN treatment, the primary change in PLUNC expression was a five-fold increase in the trachea. While the biological function of PLUNC is unknown, it is believed that it plays a role in host defense due to its antibacterial properties (Bingle and Craven, 2002Go). These real-time RT-PCR results emphasize the heterogeneity of gene expression in lung subcompartments subsequent to acute toxicant exposure and the importance of exercising caution when making conclusions regarding gene responses in difference lung microenvironments based on analysis of samples obtained from whole-lung tissue homogenates.

The greatest advantages of in situ RNA preservation combined with microdissection of lung subcompartments and RNA isolation are that: (1) RNA synthesis and degradation are immediately stopped following inflating the lungs with RNAlater, providing an immediate snapshot in time of gene expression in the lung; (2) high-quality RNA can be reproducibly isolated from identical microdissected airway segments from multiple animals; and (3) tissue microdissections and RNA isolation may be delayed without the loss of RNA integrity and quality. This technique allows investigators to measure gene expression from site-specific lung subcompartments with significant spatial and temporal precision, allowing the detection of focal changes in pulmonary gene expression that could be masked by measuring gene expression in the whole lung. Extensive detailed timecourse studies of gene expression may be performed with a high order of temporal precision using limited personnel. Previously, in order to study gene expression in such site-specific subcompartments, the lungs were first inflated with agarose at 37°C and incubated in cell culture media at 4°C, to allow the agarose to solidify, which delayed performing microdissections for 30 min (Royce et al., 1996Go). RNA from the microdissected samples needed to be isolated immediately following microdissections, and additional steps were added to the RNA isolation procedure to remove the contaminating agarose, which interfered with the RNA isolations. These requirements limited the number of animals being processed each day and are particularly problematic for inhalation exposure experiments where many animals must be necropsied at once and the number of laboratory personnel is limited. The present technique overcomes such problems and limitations. This RNA preservation and isolation technique is currently used to obtain RNA from the entire intrapulmonary airways tree for microarray analysis. To date, over 150 samples have been successfully hybridized. Some of the samples analyzed have been presented in manuscript form demonstrating differences in gene expression between the entire intrapulmonary airways tree and parenchyma (Shultz et al., 2003Go). As a result of the small amounts of RNA isolated from lung subcompartments using the present technique, we have begun incorporating RNA amplification technologies (Wang et al., 2000Go) to produce a sufficient amount of RNA for microarray analysis from lung subcompartments.

In summary, we have validated a technique that simplifies the isolation of intact, high-quality RNA from site-specific lung subcompartments that, when combined with RNA-based technologies, will significantly accelerate our understanding of the mechanisms of pulmonary injury and repair.


    ACKNOWLEDGMENTS
 
We thank Dr. Michael Evans for his critical review and suggestions in preparing this manuscript. Additionally, we thank Mr. Andrew Phimister and Ms. Jessica Reynolds for their technical assistance. This work was supported in part by NIHES04311, ES06700, ES00628, HL7013, USEPA R827442010, and the California Tobacco Related Disease Research Program of the University of California Grants TRDRP121T-0191 and 11RT-0258.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Anatomy, Physiology, and Cell Biology, 1321 Haring Hall, School of Veterinary Medicine, University of California, Davis, CA 95616. Fax: (520) 752-7690. E-mail: glbaker{at}ucdavis.edu. Back


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