Microdissected Region-specific Gene Expression Analysis with Methacarn-fixed, Paraffin-embedded Tissues by Real-time RT-PCR
Division of Pathology (HT,MS,NK,HF,K-YL,ST,MH), National Institute of Health Sciences, Tokyo; United Graduate School of Veterinary Sciences (HT,MS,KM), Gifu University, Gifu; and Laboratory of Veterinary Pathology (KM), Tokyo University of Agriculture and Technology, Tokyo, Japan
Correspondence to: Dr. M. Shibutani, Div. of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: shibutan{at}nihs.go.jp
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Summary |
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Key Words: methacarn paraffin-embedded tissue mRNA expression real-time RT-PCR microdissection hematoxylin staining
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Introduction |
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For histological assessment, tissue fixation and subsequent paraffin embedding are routinely employed because of the ease of handling tissues and subsequent staining, as well as the good preservation of morphology. Usually, formaldehyde-based fixatives, such as buffered formalin, are used for this purpose. However, with such crosslinking agents there is limited performance in terms of the yield and quality of extracted RNA (reviewed by Srinivasan et al. 2002), protein (Ikeda et al. 1998
; Shibutani et al. 2000
), and genomic DNA (Srinivasan et al. 2002
), with consequent difficulty in the analysis of microdissected, histologically defined tissue areas. Extraction efficiency and quality of molecules are critical for analysis in microdissected cells. Recently, we found that methacarn, a non-crosslinking organic solvent fixative (Puchtler et al. 1970
), meets critical criteria for analysis of RNAs, proteins, and DNAs in microdissected defined areas of paraffin-embedded tissue (PET) sections by simple extraction protocols (Shibutani et al. 2000
; Shibutani and Uneyama 2002
; Uneyama et al. 2002
). With regard to RNA expression analysis using RT-PCR, long RNA fragments as well as rare RNA species can successfully be amplified from methacarn-fixed PET sections (Shibutani et al. 2000
).
For RNA expression analysis in microdissected tissue samples, PCR-based techniques are suitable because of their sensitivity with samples having as few as 10 copies of a specific transcript. In this study we examined the suitability of methacarn fixation for measurement of mRNA expression levels in microdissected PET specimens using real-time PCR (Higuchi et al. 1992,1993
). For this purpose, we assessed (a) fidelity of mRNA expression in comparison with unfixed frozen tissue, (b) abundance of amplifiable mRNAs in comparison with unfixed cryosections, (c) linearity between the input amount of extracted total RNA and the expression level, (d) effect of tissue staining with hematoxylin, and (e) cell numbers required for practical measurement of mRNA expression in hematoxylin-stained tissue.
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Materials and Methods |
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To measure the dose-dependent induction of cytochrome P450 (CYP) 2B1 mRNA in the liver by treatment with sodium phenobarbital (PB; Wako Pure Chemical Industries, Osaka, Japan), female rats received daily IP injections of PB at doses of 0 (vehicle saline), 1.25, 5, 20, or 80 mg/kg body weight/day for 3 days and were sacrificed 24 hr after the last injection. The highest dose was selected according to the PB-specific enzyme induction protocol described by Kocarek et al. (1998). For practical assessment in microdissected areas, region-specific expression of mRNAs was measured in the hypothalamic medial preoptic area (MPOA) in male and female pups at postnatal day 10, the time point for the late stage of brain sexual differentiation in rats (Rhees et al. 1990a
,b
).
All animals used in the present study were sacrificed by exsanguination from the abdominal aorta under ether anesthesia. The animal protocols were reviewed and approved by the Animal Care and Use Committee of the National Institute of Health Sciences, Japan.
Tissue Fixation
Methacarn solution consisting of 60% (v/v) absolute methanol, 30% chloroform, and 10% glacial acetic acid was freshly prepared before fixation and stored at 4C until use. At autopsy, livers were removed and 3-mm-thick slices or 5 x 5 x 3 mm-sized tissue blocks were prepared from the left lateral lobe and fixed in methacarn for 2 hr at 4C with gentle agitation. Whole brains of rat pups were also removed and subjected to methacarn fixation. For embedding, liver slices/blocks and coronal brain slices, including the hypothalamus, were dehydrated three times for 1 hr in fresh 99.5% ethanol at 4C, immersed in xylene for 1 hr and then three times for 30 min at room temperature (RT), and immersed in hot paraffin (60C) four times for 1 hr, for a total of 4 hr. Embedded tissues were stored at 4C for up to 6 months until tissue sectioning. Unfixed liver tissue samples, either 3 x 3 x 1 mm or 5 x 5 x 3 mm, were also prepared from portions adjacent to the tissue samples for methacarn fixation and immersed in RNAlater (Ambion; Austin, TX) overnight at 4C, or embedded in Tissue-Tek 4583 OCT compound (Sakura Finetek Japan; Tokyo, Japan) by quick freezing on dry ice. They were stored at 80C until direct extraction of RNA or sectioning before RNA extraction, respectively. For immunohistochemical analysis of MPOA in pups, brains were immersed in 10% neutral buffered formalin (pH 7.4) overnight at RT with gentle agitation. Coronal brain slices that included the hypothalamus were then routinely embedded in paraffin.
Preparation of Tissue Specimens and Microdissection
For assessment of dose-dependent induction of CYP2B1 in the rat liver by PB treatment, methacarn-fixed PETs were sectioned at 10 µm and mounted on 2.5-µm PEN-foil film (Leica Microsystems; Tokyo, Japan) overlaid on a glass slide that had been treated with 3% H2O2 for 10 min, rinsed with absolute ethanol, and then dried in an incubator overnight at 37C. The sections were deparaffinized by immersion in xylene twice for 2 min, followed by 99.5% ethanol once for 30 sec. Sections were either unstained or stained with Tissue Tek Hematoxylin 3G (Sakura Finetek Japan) for 10 sec, rinsed briefly with water, and air-dried. For assessment of linearity between the input amounts of total RNA and expression levels of target genes, as well as for estimation of the relative abundance of amplifiable mRNAs, series of 20 10-µm-thick sections were prepared from 5 x 5 x 3-mm unfixed frozen tissues and methacarn-fixed PETs and were collected into 1.5-ml tubes. Integrity of extracted total RNA was also examined in these preparations by judging the resolution of rRNAs in agarose gel. In this experiment, effect of fixation itself on the integrity was also examined with fresh-frozen sections fixed with methacarn for 10 min at 4C. For preparation of tissue sections and hematoxylin staining, RNase-free ultrapure water, prefiltrated with a Gengard filter attached to an Elix 3 ultrapure water system (Millipore; Billerica, MA) was employed. Whole tissue areas of methacarn-fixed PET sections were dissected together with PEN-foil film and collected into 1.5-ml tubes for the dose-dependent expression analysis. With microdissected small tissue areas, hematoxylin-stained 10-µm-thick sections were used and circles of 30-, 50-, and 100-µm radius were microdissected from mid-zonal areas of hepatic lobules using PALM Robot-MicroBeam equipment (Carl Zeiss; Tokyo, Japan). In addition, for assessment of the relationship between the cell number and the amount of extracted total RNA, square areas of 250 x 250, 500 x 500, and 1000 x 1000 µm were also microdissected.
For microdissection of MPOA, 6-µm-thick sections between pairs of 20-µm-thick sections were prepared from methacarn-fixed rat brain PETs. The 20-µm sections were mounted on PEN-foil film. As shown in Figure 1 , localization of the sexually dimorphic nucleus of the preoptic area (SDN-POA), identified as an intensely stained cellular region, was determined under microscopic observation of 6-µm-thick sections stained with hematoxylin and eosin, and the bilateral portions of the MPOA (1000 x 600 µm) containing SDN-POA were microdissected from the adjacent unstained 20-µm-thick sections. Because of sexual dimorphism in the volume of SDN-POA, six to ten sections in males and four to six sections in females were used for microdissection.
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In cases of small tissue areas microdissected from liver PET sections, RNAqueous-Micro (Ambion) was used for total RNA extraction and the final elution volume was set at 20 µl. Contaminating genomic DNA was digested with DNase I included in the kit and the final volume was set to be 25.3 µl.
Real-time RT-PCR
When two-step real-time RT-PCR was planned, RT was performed using 1 µl (200 U) of SuperScript II RNase H Reverse Transcriptase with 2 µl of 50 µg/ml random hexamers, 1 µl of 10 mM dNTP mix, 2 µl of 10 x PCR buffer, 1.2 µl of 50 mM MgCl2, 2 µl of 0.1 M dithiothreitol, 1 µl of RNase inhibitor, and 9.8 µl of RNA solution in a 20-µl total reaction volume (all reagents were purchased from Invitrogen; Carlsbad, CA). After treatment with 1 µl of RNase H, 1 µl of RT product was subjected to real-time PCR in a 25 µl of total reaction volume with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems Japan; Tokyo, Japan) using either QuantiTect SYBR Green PCR Kit (QIAGEN) or TaqMan Universal PCR Master Mix (Applied Biosystems Japan). With this two-step RT-PCR, mRNA expression levels of CYP2B1, estrogen receptor (ER), ERß,
-aminobutyric acid transporter type 1 (GAT-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were measured. Primer Express software (Version 2.0; Applied Biosystems Japan) was used for the design of primer sequences and TaqMan probes. For expression analysis of GAPDH, either SYBR Green or TaqMan probe system was applied. In the latter case, TaqMan Rodent GAPDH Control Reagents (Applied Biosystems Japan) were used. The sequences of primers and probes are listed in Table 1.
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When RT and following real-time PCR were intended to be performed sequentially in one tube, one-step kits, such as the QuantiTect SYBR Green RT-PCR Kit (QIAGEN; for CYP2B1) and the QuantiTect Probe RT-PCR Kit (QIAGEN; for GAPDH) were used with 5 µl of total RNA in a 50-µl total reaction volume according to the manufacturer's protocols. Cycle parameters for CYP2B1 were similar to the above described two-step case, and a RT step at 50C for 30 min was preceded for the initial activation step at 95C for 10 min. In the case of GAPDH, cycle parameters were as follows: single step of 50C for 30 min; single step of 95C for 15 min; and 50 cycles of 94C for 15 sec followed by 60C for 60 sec.
As a negative control for RT, reverse transcriptase () mock RT samples were included in each PCR experiment.
Immunohistochemical Analysis
Because ER mRNA is differentially expressed in the MPOA depending on the gender, the corresponding protein expression was also examined immunohistochemically. A series of five 3-µm-thick sections were prepared at 30-µm intervals through the MPOA and the first of each series was stained with hematoxylin and eosin. These sections were examined microscopically and one showing the maximum size of SDN-POA was identified and selected for IHC with ER
in each animal. Deparaffinized and hydrated sections were treated with microwaving for 9 min in 0.01 M citrate buffer (pH 6.0) and treated with 1% periodic acid solution for 10 min. After incubation with mouse anti-ER
monoclonal antibody (Novocastra Laboratories, Newcastle upon Tyne, UK; x 40 dilution), immunodetection was performed using a VECTASTAIN Elite ABC KIT (Vector Laboratories; Burlingame, CA) with a standard protocol using diaminobenzidine as chromogen. The sections were then counterstained with hematoxylin. Digital photomicrographs at a magnification of x180 were taken with a Fujix Digital Camera system (Fujifilm; Tokyo, Japan), and the numbers of immunostained nuclei within the MPOA (600 x 1000-µm areas) were counted using MacSCOPE (version 2.65; Mitani, Fukui, Japan).
Statistical Analysis
Comparison of data of mRNA expression levels and ER-immunoreactive cell numbers in MPOA was performed with the Student's t-test after confirmation of equal variance of values. Pearson's correlation coefficients were calculated between the input amounts of RNA and the target gene expression levels in the validation studies in the liver and MPOA. Variability was expressed as coefficient of variation (CV).
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Results |
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Discussion |
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Generally speaking, tissue staining with hematoxylin appears to affect both extraction efficiency and PCR amplification of genomic DNA (Murase et al. 2000; Serth et al. 2000
), although in the present study the influence of brief staining with hematoxylin was marginal. Polynucleotides in tissue sections fixed with organic solvent fixatives, such as Carnoy's solution or methacarn, may be released into solution during IHC or ISH (Urieli-Shoval et al. 1992
; Uneyama et al. 2002
). However, methacarn-fixed PET sections could quickly be stained with hematoxylin in a period of 110 sec (Uneyama et al. 2002
), and this might have contributed to the limited loss of extractable RNA after hematoxylin staining in the present study. We also observed only slight reduction (020%) in the relative abundance of amplifiable mRNAs after hematoxylin staining. We previously noted that staining with hematoxylin and eosin affected PCR of genomic DNA in methacarn-fixed PET sections, despite no deterioration of the integrity and yield of extracted DNA (Uneyama et al. 2002
). Hematoxylin has been reported to influence divalent cations (Mg2+) that are important for maintaining Taq DNA polymerase activity (Chen et al. 1996
), and this was reported to be apparent when manually dissected large tissue samples are subjected to PCR analysis (Burton et al. 1998
; Murase et al. 2000
). However, such an inhibitory effect might be negligible when microdissected small tissue specimens are analyzed (Ehrig et al. 2001
). In line with the present study results, hematoxylin staining did not affect RT-PCR when microdissected small tissue specimens were analyzed in a previous study (Imamichi et al. 2001
).
The cell number required for mRNA expression analysis in microdissected tissue specimens is primarily dependent on the expression level of the target genes of interest. In the case of cyclin D1 in primary tumor tissues, Specht et al. (2002) reported that transcripts could be measured in a minimum of 20 microdissected tumor cells from formalin-fixed PET specimens with an improved extraction protocol and the TaqMan PCR method in combination, but they also found 2000 cells to be suitable to obtain reproducible real-time PCR results. Although the expression values did not greatly vary (CV <20) from 100 pg of total RNA in the present experiment examining relative abundance of amplifiable mRNAs (Table 2),
200 cells (corresponding to 2 ng based on the RNA yield data in Table 5) can be considered as a minimum for the practical expression analysis of mRNA species, judging from the very small variation of difference in CT values of GAPDH gene with the corresponding tissue size (100 µm in radius x 4 pieces) reflecting homogeneity in the expression between samples as well as very small technical variation (Table 4). Expression levels of CYP2B1 varied between samples, even with a tissue area of 208 cells. With PB treatment, graded expression of CYP2B1 in the liver lobule occurs in the rat, with pronounced induction in the periportal region (Bühler et al. 1992
). In the present study, mid-zonal areas of hepatic lobules were subjected to analysis, and therefore the variability of CYP2B1 expression in each sample might rather reflect a local event due to the graded expression profile of the transcript within this area.
In the present study, the concentration of total RNA was measured by RiboGreen fluorescent dye, with 1 ng as the lower detection limit in a 1-ml assay volume, and we could obtain 1 ng of total RNA from 100 microdissected liver cells. If normalization of mRNA expression level to the input amount of total RNA is intended, a total of 3 ng or more (corresponding to >300 cells in a 10-µm-thick section of the rat liver) would be necessary for measurement of RNA concentration (1 ng) and after real-time RT-PCR of several genes (2 ng) under the present experimental conditions. If a fluorescence microplate reader is available, the total volume for assay of total RNA concentration could be minimized. As another normalization method, the expression level of a reference gene can be used. Housekeeping genes, such as GAPDH in the present study, are usually selected for this purpose. However, disadvantages with a single housekeeping gene were recently reported (Lee et al. 2002
; Tricarico et al. 2002
), and sexual dimorphism exists in GAPDH expression in several brain regions of developing rats (Perrot-Sinal et al. 2001
). Sexual dimorphism of ER
expression in MPOA was apparent here, irrespective of the normalization method.
In conclusion, we have now demonstrated that methacarn-fixed PET allows practical mRNA expression analysis in microdissected areas with real-time RT-PCR after hematoxylin staining. Although recent studies have also demonstrated good performance of mRNA expression measurement with microdissected formalin-fixed PETs (Specht et al. 2001,2002
), formaldehyde causes modification of nucleotides and therefore a high frequency of non-reproducible sequence alteration and amplification of only short fragments (reviewed by Srinivasan et al. 2002
), suggesting limited utility for molecular analysis. Considering the availability for both DNAs and proteins in PET sections (Shibutani et al. 2000
; Shibutani and Uneyama 2002
; Uneyama et al. 2002
), methacarn should prove to be a versatile tool for multipurpose analysis of target genes in specific cell populations. We are now applying methacarn for global gene expression analysis using a microarray technique with microdissected PET specimens. The question of how long molecules are retained intact in methacarn-fixed PET should now be addressed. Although we do not have data for archival tissues stored for several years/decades, mRNA levels could be measured with methacarn-fixed PET that had been prepared 6 months previously in the present study.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Bühler R, Lindros KO, Nordling Å, Johansson I, Ingelman-Sundberg M (1992) Zonation of cytochrome P450 isozyme expression and induction in rat liver. Eur J Biochem 204:407412[Abstract]
Burton MP, Schneider BG, Brown R, Escamilla-Ponce N, Gulley ML (1998) Comparison of histologic stains for use in PCR analysis of microdissected, paraffin-embedded tissues. Biotechniques 24:8692[Medline]
Chen JT, Lane MA, Clark DP (1996) Inhibitors of the polymerase chain reaction in Papanicolaou stain. Removal with a simple destaining procedure. Acta Cytol 40:873877[Medline]
Ehrig T, Abdulkadir SA, Dintzis SM, Milbrandt J, Watson MA (2001) Quantitative amplification of genomic DNA from histological tissue sections after staining with nuclear dyes and laser capture microdissection. J Mol Diagn 3:2225
Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, et al. (1996) Laser capture microdissection. Science 274:9981001
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology 10:413417[Medline]
Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:10261030[Medline]
Ikeda K, Monden T, Kanoh T, Tsujie M, Izawa H, Haba A, Ohnishi T, et al. (1998) Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections. J Histochem Cytochem 46:397403
Imamichi Y, Lahr G, Wedlich D (2001) Laser-mediated microdissection of paraffin sections from Xenopus embryos allows detection of tissue-specific expressed mRNAs. Dev Genes Evol 211:361366[CrossRef][Medline]
Kocarek TA, Kraniak JM, Reddy AB (1998) Regulation of rat hepatic cytochrome P450 expression by sterol biosynthesis inhibition: inhibitors of squalene synthase are potent inducers of CYP2B expression in primary cultured rat hepatocytes and rat liver. Mol Pharmacol 54:474484
Lee PD, Sladek R, Greenwood CM, Hudson TJ (2002) Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res 12:292297
Murase T, Inagaki H, Eimoto T (2000) Influence of histochemical and immunohistochemical stains on polymerase chain reaction. Mod Pathol 13:147151[CrossRef][Medline]
Orikasa C, Kondo Y, Hayashi S, McEwen BS, Sakuma Y (2002) Sexually dimorphic expression of estrogen receptor ß in the anteroventral periventricular nucleus of the rat preoptic area: implication in luteinizing hormone surge. Proc Natl Acad Sci USA 99:33063311
Perrot-Sinal TS, Davis AM, McCarthy MM (2001) Developmental sex differences in glutamic acid decarboxylase (GAD(65)) and the housekeeping gene, GAPDH. Brain Res 922:201208[CrossRef][Medline]
Puchtler H, Waldrop FS, Meloan SN, Terry MS, Conner HM (1970) Methacarn (methanol-Carnoy) fixation. Practical and theoretical considerations. Histochemie 21:97116[Medline]
Rhees RW, Shryne JE, Gorski RA (1990a) Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats. J Neurobiol 21:781786[Medline]
Rhees RW, Shryne JE, Gorski RA (1990b) Termination of the hormone-sensitive period for differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats. Dev Brain Res 52:1723[Medline]
Schütze K, Lahr G (1998) Identification of expressed genes by laser-mediated manipulation of single cells. Nature Biotechol 16:737742[Medline]
Serth J, Kuczyk MA, Paeslack U, Lichtinghagen R, Jonas U (2000) Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections. Am J Pathol 156:11891196
Shibutani M, Uneyama C, Miyazaki K, Toyoda K, Hirose M (2000) Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens. Lab Invest 80:199208[Medline]
Shibutani M, Uneyama C (2002) Methacarn: a fixation tool for multipurpose genetic analysis from paraffin-embedded tissues. In Conn PM, ed. Laser Capture Microscopy and Microdissection. Methods Enzymol 356. London, Academic Press, 114125
Specht K, Kremer M, Muller U, Dirnhofer S, Rosemann M, Hofler H, Quintanilla-Martinez L, Fend F (2002) Identification of cyclin D1 mRNA overexpression in B-cell neoplasias by real-time reverse transcription-PCR of microdissected paraffin sections. Clin Cancer Res 8:29022911
Specht K, Richter T, Muller U, Walch A, Werner M, Hofler H (2001) Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue. Am J Pathol 158:419429
Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161:19611971
Tricarico C, Pinzani P, Bianchi S, Paglierani M, Distante V, Pazzagli M, Bustin SA, et al. (2002) Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal Biochem 309:293300[CrossRef][Medline]
Uneyama C, Shibutani M, Masutomi N, Takagi H, Hirose M (2002) Methacarn fixation for genomic DNA analysis in microdissected, paraffin-embedded tissue specimens. J Histochem Cytochem 50:12371245
Urieli-Shoval S, Meek RL, Hanson RH, Ferguson M, Gordon D, Benditt EP (1992) Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation. J Histochem Cytochem 40:18791885
Yokosuka M, Okamura H, Hayashi S (1997) Postnatal development and sex difference in neurons containing estrogen receptor-alpha immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat. J Comp Neurol 389:8193[CrossRef][Medline]