APMIS 100: 498-502, 1992
Northern and Southern blot analysis of human RNA and DNA in autopsy material SUSANNE LARSENI, f i R E RYGAARD', STIG ASNiES2 and MOGENS SPANG-THOMSEN' 'Institute of Pathological Anatomy, and 21nstituteof Forensic Medicine, University of Copenhagen, Copenhagen, Denmark
Larsen, S., Rygaard, K., Asnies, S. & Spang-Thomsen, M. Northern and Southern blot analysis of human RNA and DNA in autopsy material. APMIS ZOO: 498-502, 1992. Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed 21-118 h postmortem. Extracted RNA and DNA were examined by Northern and Southern blot analysis using oligolabelled human DNA probes recognizing gene transcripts of 2-5 kb. The results indicated that, in general, Northern blot analysis was feasible with the applied probes when the tissue was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five days postmortem, depending on the organ from which the DNA was extracted. Key words: RNA; DNA; autopsy; Northern blotting; Southern blotting. Susanne Larsen, Institute of Pathological Anatomy, University of Copenhagen, 11 Frederik V Vej, DK-2 100 Copenhagen, Denmark.
In molecular biological investigations there is sometimes a need for reference material from normal human tissues, but fresh biopsy material may not be obtainable from all relevant tissues, e.g. brain and heart. In this study the feasibility of using RNA and DNA isolated from autopsy material in Northern and Southern blot analysis was investigated. The results showed that RNA was generally sufficiently well preserved to obtain signals on Northern blots with the probes applied when the tissue was obtained less than two days postmortem. Histological examination showing only slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative of preservation of RNA. Southern
Received April 24, 1991. Accepted December 2, 1991.
498
blot analysis of tissue was feasible at less than three to five days postmortem, depending on the organ from which the DNA was extracted.
MATERIALS AND METHODS Tissue sampling Tissue samples were obtained from 10 autopsies performed 21-118 h postmortem at the University Institute of Forensic Medicine, Copenhagen and at the Department of Pathology, Rigshospitalet, Copenhagen. Autopsies performed more than five days postmortem or with obvious signs of putrefaction were excluded. In order to obtain samples from undisturbed tissue and to avoid possible differences in gene expression between adults and children, patients with a history of malignancy or intravenous drug abuse, and those aged under 20 years were excluded. Representative samples were taken from brain, heart, lung, liver, spleen, kidney and adrenal glands, and snap
RNA AND DNA FROM AUTOPSIES
frozen in liquid nitrogen. A part of each sample was prepared for routine histological examination. Degradation of mRNA under controlled conditions was studied in a separate experiment, using a human small cell lung cancer (SCLC) nude mouse xenograft GLC-19 (Berendsen et d.1988) as the tissue source. A total of 12 xenografts measuring 4 1 3 mm were used. Tumours were excised, freed from necrosis, and cut into pieces. One piece from each tumour was immediately snap frozen in liquid nitrogen, and the remaining pieces were kept in phosphate-buffered saline at 4OCkO.2 and 2OoC+O.2, and snap frozen at intervals from 20 min to 72 h after excision. Each sampling included pieces from all 12 tumours, and these 12 tumour pieces were pooled into three samples, each thus representing four of the 12 tumours. RNA analysis of these triplicate samples was performed separately on each individual sample. Histology The degree of autolysis was histologically grouped into five categories: none, none-slight, slight, moderate, and heavy. RNA and D N A extraction RNA was isolated from the autopsy tissues and the GLC-19 tumour samples by the acid guanidinium thiocyanate-phenol-chloroformmethod (Chomczynski & Sacchi 1987), and the concentration was determined by spectrophotometry. DNA was isolated from autopsy tissue samples by standard phenol-chloroform extraction (Sumbrook et al. 1989). The concentration of DNA was determined by fluorometry (Labarca & Paigen 1980). Northern and Southern blotting RNA was dissolved in loading buffer containing ethidium bromide. Ten pg of each sample was size fractionated through 1% formaldehyde-agarose gels, and transferred to nylon membranes (Genescreen Plus, NEN Dupont). DNA was digested enzymatically with the restriction endonuclease EcoRI (Bethesda Research Laboratories). Ten pg DNA samples were electrophoresed through 0.8% agarose gels, and transferred to Genescreen Plus membranes. The membranes were prehybridized in a solution containing 50% formamide, 1% sodium dodecyl sulfate (SDS), 1M NaCl, 5% dextran sulfate, and 100 pg/ml salmon testis DNA at 4 2 T , and hybridized to relevant human DNA probes for 16-18 h at 42°C. The membranes were washed with a washing stringency of 2xSSC, 1% SDS, twice for 30 min at 60-65°C. The membranes were exposed to X-ray films with an intensifying screen at -80°C for one to seven days. The probe for the retinoblastoma gene was a 3.8 kb EcoRI fragment of the cloned 4.7 kb cDNA (Friend et ul. 1986), the c-ruf-1 probe was a 2.0 kb EcoRI-PvuII
fragment of the plasmid p627 (Bonner et al. 1985), the c-myc probe was a 0.9 kb EcoRI-CZaI fragment of the plasmid pHSR-1 (AZitalo et al. 1983), and the p-actin probe was a 2.1 kb BamHI fragment of the plasmid pHFPA-1 (Gunning et al. 1983). ["PI-radiolabelled probes were prepared by the random priming method (Feinberg & Vogelstein 1983) using a commercial kit (Amersham). All RNA samples were probed for p-actin gene transcripts. RNA from the autopsies was probed for retinoblastoma gene transcripts, and RNA samples from the GLC-19 tumour xenograft were probed for c-ruf-1 and c-myc transcripts, because the tumour was known to express these genes at the mRNA level. The DNA samples were probed for the retinoblastoma gene.
RESULTS Histology Examination of the histological sections confirmed that none of the samples contained malignant tissue. All the organs of six out of the 10 autopsies showed none-slight to moderate autolysis. In the organs of the remaining four autopsies the autolysis was generally heavy, with the exception that in all brain and in two spleen samples autolysis was only slight.
ribosomal bands
4.7 -
RB -
2.0 -
p-actin
Fig. 1. Ethidium bromide-stained agarose gel after electrophoretic size fractionation of the RNA (top), and the corresponding Northern blot analysis of ten autopsy lung samples probed for the human p-actin and retinoblastoma gene transcripts (bottom). Two ribosomal bands appeared in the gels when the RNA was undegraded. Northern blot signals of different intensity were obtained in 7/ 10 lung samples examined. The molecular weight of transcripts was determined with reference to the 28s and 18s rRNA bands. The postmortem sampling times are indicated.
499
LARSEN et al.
Northern blotting The results of the Northern blotting of RNA from autopsy material are summarized in Table 1. Ribosomal RNA bands could generally be visualized on the ethidium bromide-stained gels when the tissue was sampled from autopsies performed I 50 h postmortem. In contrast, with few exceptions the ribosomal bands were absent in autopsies performed >50 h postmortem, indicating that in these tissues the RNA was degraded. Correspondingly, probing for the retinoblastoma and p-actin transcripts was generally only successful in the cases where the ribosomal bands could be demonstrated. In this group (I50 h postmortem), the seven spleen samples differed from the general pattern: only two and three samples hybridized to the retinoblastoma and P-actin probes, respectively. The RNA results in Fig. 1 show the agarose gel and Northern blots of the lung samples probed for the retinoblastoma gene transcripts. It appears that when two ribosomal bands were visible on the gels, signals were obtained on Northern blots.
Heart
Southern blotting The Southern blot analysis (Table 1) showed that generally DNA from autopsies performed 2 7 5 h postmortem hybridized to the retinoblastoma gene probe. However, in lung, adrenal glands, and brain, hybridization was successful even in tissue from autopsies performed five days postmortem. Fig. 2 illustrates the Southern blot analysis for the adrenal gland samples after hybridization to the retinoblastoma gene probe.
Xenograjl study The results of the RNA degradation study at 20°C are illustrated in Fig. 3. The ribosomal RNA bands and Northern blot signals were present and undegraded until 24 h after excision, were fading at 48 h, and had almost disappeared at 72 h. In the samples from tissue kept at 4"C, ribosomal bands and mRNA signals were similar at all time points investigated (data not shown).
TABLE I. RNA and DNA analvsis o f normal human tissues from I0 automies RNA analysis DNA analysis Postmortem Ribosomal Rb p-actin Postmortem Rb time fh) bands time (h) 5 50 818 718 618 I75 819
> 50
012 618 212 618 112 517 012 517 012 If 7 012 415 112
012 618 112 518 012 217 012 517 012 717 012 515 1/ 2
012 618 1/ 2 618 012 317 012 517 012 717 012 515 112
> 75
01 1 617
I50 5 75 > 50 > 75 111 Liver I 50 < 75 919 > 50 75 01 1 Spleen2) 5 50 I75 618 > 50 > 75 01 1 Kidney2) I50 < 75 818 > 50 575 01 1 I 50 I75 818 Ad rena 1 > 50 > 75 1/1 gland') Brain3) I 50 I75 616 > 50 > 75 11 1 The RNA samples were examined for the presence of ribosomal bands after size fractionation on the ethidiumstained gels, and were probed for the retinoblastoma (Rb) and p-actin transcripts. The extracted DNA was probed for the Rb gene. ') In two lung samples the DNA yield was insufficient for Southern blot analysis. )' The spleen, kidneys, and adrenal glands had been removed from one autopsy. )) Brain tissue was not available in three autopsies.
Lung')
500
r
RNA A N D DNA FROM AUTOPSIES
23.1 -
*
9.4 -
6.6-
4.4 -
2.3-
Fig. 2. Southern blot analysis of seven autopsy adrenal gland samples probed for the human retinoblastoma gene, DNA was digested with the restriction endonuclease EcoRI. Signals of varying intensity were obtained from all samples. HindII-digested hphage DNA was used as size marker. The postmortem sampling times are indicated.
DISCUSSION The RNA analyses of the autopsy and xenograft tissues were in good agreement, indicating that mRNA was appropriate for Northern blot analysis with the applied probes for approximately 50 h postmortem. Furthermore, the xenograft results suggested that this period may be extended if the tissue (corpse) is kept at lower temperatures.
1-
2.32.0
-
I ,
c-myc I
p-actin
Fig. 3. The agarose gel and corresponding Northern blot analysis of samples from a small cell lung cancer xenograft kept at 20°C from 0 to 72 h after excision. The blots were hybridized to probes for the c-ruf-1, c-myc, and p-actin transcripts. The molecular weight of transcripts was determined with reference to the 28s and 18s rRNA bands.
Histological examination showing only slight or no autolysis and the presence of ribosomal RNA after gel electrophoresis were indicative of the presence in the autopsy samples of mRNA appropriate for Northern blot analysis with the applied probes. The present investigations were carried out with probes recognizing gene transcripts of 2.1 to 4.7 kb and thus covering the size range of a large proportion of mRNAs. However, it should be emphasized that gene transcripts larger than that of the Rb gene (4.7 kb) may not be preserved to the same degree. Furthermore, the results showed only that autopsy material may be used for qualitative detection of gene transcripts. Partial degradation may have caused a decrease in the levels of expression as indicated by the different signal intensities obtained (Figs. 1 & 2). In other studies, mRNA was isolated from lung until 20 h (Hymphreys-Beher et al. 1986), from liver until 16 h (Finger et al. 1987), and from brain tissue until 36 h postmortem (Wood et al. 1986, Johnson et al. 1986, Morrison & GrijBn 1981). We detected RNA for longer periods in all tissues examined (Table 1). The xenograft experiment demonstrated that the RNA stability was strongly dependent on the temperature. The discrepancy between the present and other results might thus be ascribed to different conditions of postmortem tissue storage. As for RNA, the degree of DNA degradation was found to be dependent on the time lapse between death and autopsy (Table 1). However, DNA was generally preserved for longer periods, and the variation in stability among the organs examined was greater for DNA than for RNA. In all organs examined DNA was detected until three days postmortem, but in several cases DNA was isolated from tissues sampled five days postmortem. These findings are in fair agreement with the reported postmortem stability of 2 4 3 6 h in liver, five days in spleen, kidney and thyroid, and even longer in brain (Bur et al. 1988). In contrast to the RNA findings, DNA stability was not correlated with the histological grading of tissue autolysis. Several DNA samples from tissue graded as heavily autolysed provided a signal by Southern blot analysis. The results with respect to brain tissue repre50 1
LARSEN ef al.
sented a distinct divergence from the general pattern. Irrespective of the histological grading of the other organs, all brain samples showed only slight to moderate autolysis, and signals were obtained in almost all Northern and Southern blot analyses (Table l), independent of the postmortem sampling time. This is in concordance with the reported high frequency of isolation of undegraded DNA from autopsy brain tissue (Upadhyaya et al. 1985). In the xenograft investigation, there was a parallel variation in signal intensity with the three probes employed (Fig. 3). This was rather unexpected in view of the short in vivo half-life of the c-myc mRNA (10-20 min) (Dani et al. 1984) compared to the approximately 6-12 h reported for P-actin (Khalili& Weinmann 1984). One explanation for this extended c-myc mRNA stability could be a reduced cellular metabolic rate postmortem. Alternatively, the cells sustained unchanged mRNA production for two days following clinical death. This study was supported by the Weimann Foundation, the Michaelsen Foundation, and the Danish Cancer Society. The technical assistance of Jette R~hrmannis greatly appreciated.
REFERENCES Alitalo, K., Schwab, M . , Lin, C. C,,Varmus, H. E. & Bishop, J. M .:Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc. Natl. Acad. Sci. USA 80: 1707-171 I , 1983. Berendsen, H. H., de Leij, L., de Vries, E. G. E., Mesander, G., Mulder, N . H., de Jong, B., Buys, C. H. C.M., Postmus, I? E., Poppema, S., Sluiter, H. J. & The, H. T : Characterization of three small cell lung cancer cell lines established from one patient during longitudinal follow-up. Cancer Res. 48: 6891-6899, 1988. Bonner, T. I., Kerby, S. B., Sutrave, P,Gunnell, M . A., Mark, G. & Rapp, U. R. Structure and biological activity of human homologs of the raf/mil oncogene. Mol. Cell Biol. 5: 1400-1407, 1985. Bar, W , Kratzer, A., Machler, M. & Schmid, W: Postmortem stability of DNA. Forensic Sci. Int. 39: 59-70, 1988. Chomczynski, I! & Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-
502
phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. Dani, C. H., Blanchard, J. M., Piechaczyk, M., El Sabouty, S., Murty, L. & Jeanteur, I!: Extreme instability of myc mRNA in normal and transformed human cells. Proc. Natl. Acad. Sci. USA 81: 70467050, 1984. Feinberg, A. I! & Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13, 1983. Finger, J. M . , Mercer, J. I? B., Cotton, R. G. H. & Danks, D. M.: Stability of protein and mRNA in human postmortem liver - analysis by twodimensional gel electrophoresis. Clin. Chim. Acta 170: 209-218, 1987. Friend, S. H., Bernards, R., Rogelj, S., Weinberg, R. A., Rapaport, J. M , , Albert, D. M . & Dryja, T I?: A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323: 642-646, 1986. Gunning, P,Ponte, I!, Okayama, H., Engel, J., Blau. H. & Kedes, L.: Isolation and characterization of full-length cDNA clones for human a-, p- and yactin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol. Cell Biol. 3: 787-795, 1983. Humphreys-Beher, M . G., King, I? K., Bunnel, B. & Brody, B.: Isolation of biologically active RNA from human autopsy for the study of cystic f i brosis. Biotechnol. Appl. Biochem. 8: 392403, 1986. Johnson, S. A., Morgan, D. G. & Finch, C.E.: Extensive postmortem stability of RNA from rat and human brain. J. Neurosci. Res. 16: 267-280, 1986. Khalili, K . & Weinmann, R.: Actin mRNAs in HeLa cells. Stabilization after adenovirus infection. J. Mol. Biol. 180: 1007-1021, 1984. Labarca, C. & Paigen, K.: A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102: 344-352, 1980. Morrison, M . R. & Griffin, S. T: The isolation and in vitro translation of undegraded messenger RNAs from human postmortem brain. Anal. Biochem. 113: 318-324, 1981. Sambrook, J., Fritsch, E. I? & Maniatis, T : Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Upadhyaya, M., Reynolds, G. I! & Harper, I! S.: Recombinant DNA studies on stored necropsy brain samples from patients with‘ Huntington’s chorea. J. Clin. Pathol. 38: 1093-1095, 1985. Wood, T L., Frantz, G. D., Menkes, J . H. & Tobin, A. J.: Regional distribution of messenger RNAs in postmortem human brain. J. Neurosci. Res. 16: 311-324, 1986.
Northern and Southern blot analysis of human RNA and DNA in autopsy material SUSANNE LARSENI, f i R E RYGAARD', STIG ASNiES2 and MOGENS SPANG-THOMSEN' 'Institute of Pathological Anatomy, and 21nstituteof Forensic Medicine, University of Copenhagen, Copenhagen, Denmark
Larsen, S., Rygaard, K., Asnies, S. & Spang-Thomsen, M. Northern and Southern blot analysis of human RNA and DNA in autopsy material. APMIS ZOO: 498-502, 1992. Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed 21-118 h postmortem. Extracted RNA and DNA were examined by Northern and Southern blot analysis using oligolabelled human DNA probes recognizing gene transcripts of 2-5 kb. The results indicated that, in general, Northern blot analysis was feasible with the applied probes when the tissue was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five days postmortem, depending on the organ from which the DNA was extracted. Key words: RNA; DNA; autopsy; Northern blotting; Southern blotting. Susanne Larsen, Institute of Pathological Anatomy, University of Copenhagen, 11 Frederik V Vej, DK-2 100 Copenhagen, Denmark.
In molecular biological investigations there is sometimes a need for reference material from normal human tissues, but fresh biopsy material may not be obtainable from all relevant tissues, e.g. brain and heart. In this study the feasibility of using RNA and DNA isolated from autopsy material in Northern and Southern blot analysis was investigated. The results showed that RNA was generally sufficiently well preserved to obtain signals on Northern blots with the probes applied when the tissue was obtained less than two days postmortem. Histological examination showing only slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative of preservation of RNA. Southern
Received April 24, 1991. Accepted December 2, 1991.
498
blot analysis of tissue was feasible at less than three to five days postmortem, depending on the organ from which the DNA was extracted.
MATERIALS AND METHODS Tissue sampling Tissue samples were obtained from 10 autopsies performed 21-118 h postmortem at the University Institute of Forensic Medicine, Copenhagen and at the Department of Pathology, Rigshospitalet, Copenhagen. Autopsies performed more than five days postmortem or with obvious signs of putrefaction were excluded. In order to obtain samples from undisturbed tissue and to avoid possible differences in gene expression between adults and children, patients with a history of malignancy or intravenous drug abuse, and those aged under 20 years were excluded. Representative samples were taken from brain, heart, lung, liver, spleen, kidney and adrenal glands, and snap
RNA AND DNA FROM AUTOPSIES
frozen in liquid nitrogen. A part of each sample was prepared for routine histological examination. Degradation of mRNA under controlled conditions was studied in a separate experiment, using a human small cell lung cancer (SCLC) nude mouse xenograft GLC-19 (Berendsen et d.1988) as the tissue source. A total of 12 xenografts measuring 4 1 3 mm were used. Tumours were excised, freed from necrosis, and cut into pieces. One piece from each tumour was immediately snap frozen in liquid nitrogen, and the remaining pieces were kept in phosphate-buffered saline at 4OCkO.2 and 2OoC+O.2, and snap frozen at intervals from 20 min to 72 h after excision. Each sampling included pieces from all 12 tumours, and these 12 tumour pieces were pooled into three samples, each thus representing four of the 12 tumours. RNA analysis of these triplicate samples was performed separately on each individual sample. Histology The degree of autolysis was histologically grouped into five categories: none, none-slight, slight, moderate, and heavy. RNA and D N A extraction RNA was isolated from the autopsy tissues and the GLC-19 tumour samples by the acid guanidinium thiocyanate-phenol-chloroformmethod (Chomczynski & Sacchi 1987), and the concentration was determined by spectrophotometry. DNA was isolated from autopsy tissue samples by standard phenol-chloroform extraction (Sumbrook et al. 1989). The concentration of DNA was determined by fluorometry (Labarca & Paigen 1980). Northern and Southern blotting RNA was dissolved in loading buffer containing ethidium bromide. Ten pg of each sample was size fractionated through 1% formaldehyde-agarose gels, and transferred to nylon membranes (Genescreen Plus, NEN Dupont). DNA was digested enzymatically with the restriction endonuclease EcoRI (Bethesda Research Laboratories). Ten pg DNA samples were electrophoresed through 0.8% agarose gels, and transferred to Genescreen Plus membranes. The membranes were prehybridized in a solution containing 50% formamide, 1% sodium dodecyl sulfate (SDS), 1M NaCl, 5% dextran sulfate, and 100 pg/ml salmon testis DNA at 4 2 T , and hybridized to relevant human DNA probes for 16-18 h at 42°C. The membranes were washed with a washing stringency of 2xSSC, 1% SDS, twice for 30 min at 60-65°C. The membranes were exposed to X-ray films with an intensifying screen at -80°C for one to seven days. The probe for the retinoblastoma gene was a 3.8 kb EcoRI fragment of the cloned 4.7 kb cDNA (Friend et ul. 1986), the c-ruf-1 probe was a 2.0 kb EcoRI-PvuII
fragment of the plasmid p627 (Bonner et al. 1985), the c-myc probe was a 0.9 kb EcoRI-CZaI fragment of the plasmid pHSR-1 (AZitalo et al. 1983), and the p-actin probe was a 2.1 kb BamHI fragment of the plasmid pHFPA-1 (Gunning et al. 1983). ["PI-radiolabelled probes were prepared by the random priming method (Feinberg & Vogelstein 1983) using a commercial kit (Amersham). All RNA samples were probed for p-actin gene transcripts. RNA from the autopsies was probed for retinoblastoma gene transcripts, and RNA samples from the GLC-19 tumour xenograft were probed for c-ruf-1 and c-myc transcripts, because the tumour was known to express these genes at the mRNA level. The DNA samples were probed for the retinoblastoma gene.
RESULTS Histology Examination of the histological sections confirmed that none of the samples contained malignant tissue. All the organs of six out of the 10 autopsies showed none-slight to moderate autolysis. In the organs of the remaining four autopsies the autolysis was generally heavy, with the exception that in all brain and in two spleen samples autolysis was only slight.
ribosomal bands
4.7 -
RB -
2.0 -
p-actin
Fig. 1. Ethidium bromide-stained agarose gel after electrophoretic size fractionation of the RNA (top), and the corresponding Northern blot analysis of ten autopsy lung samples probed for the human p-actin and retinoblastoma gene transcripts (bottom). Two ribosomal bands appeared in the gels when the RNA was undegraded. Northern blot signals of different intensity were obtained in 7/ 10 lung samples examined. The molecular weight of transcripts was determined with reference to the 28s and 18s rRNA bands. The postmortem sampling times are indicated.
499
LARSEN et al.
Northern blotting The results of the Northern blotting of RNA from autopsy material are summarized in Table 1. Ribosomal RNA bands could generally be visualized on the ethidium bromide-stained gels when the tissue was sampled from autopsies performed I 50 h postmortem. In contrast, with few exceptions the ribosomal bands were absent in autopsies performed >50 h postmortem, indicating that in these tissues the RNA was degraded. Correspondingly, probing for the retinoblastoma and p-actin transcripts was generally only successful in the cases where the ribosomal bands could be demonstrated. In this group (I50 h postmortem), the seven spleen samples differed from the general pattern: only two and three samples hybridized to the retinoblastoma and P-actin probes, respectively. The RNA results in Fig. 1 show the agarose gel and Northern blots of the lung samples probed for the retinoblastoma gene transcripts. It appears that when two ribosomal bands were visible on the gels, signals were obtained on Northern blots.
Heart
Southern blotting The Southern blot analysis (Table 1) showed that generally DNA from autopsies performed 2 7 5 h postmortem hybridized to the retinoblastoma gene probe. However, in lung, adrenal glands, and brain, hybridization was successful even in tissue from autopsies performed five days postmortem. Fig. 2 illustrates the Southern blot analysis for the adrenal gland samples after hybridization to the retinoblastoma gene probe.
Xenograjl study The results of the RNA degradation study at 20°C are illustrated in Fig. 3. The ribosomal RNA bands and Northern blot signals were present and undegraded until 24 h after excision, were fading at 48 h, and had almost disappeared at 72 h. In the samples from tissue kept at 4"C, ribosomal bands and mRNA signals were similar at all time points investigated (data not shown).
TABLE I. RNA and DNA analvsis o f normal human tissues from I0 automies RNA analysis DNA analysis Postmortem Ribosomal Rb p-actin Postmortem Rb time fh) bands time (h) 5 50 818 718 618 I75 819
> 50
012 618 212 618 112 517 012 517 012 If 7 012 415 112
012 618 112 518 012 217 012 517 012 717 012 515 1/ 2
012 618 1/ 2 618 012 317 012 517 012 717 012 515 112
> 75
01 1 617
I50 5 75 > 50 > 75 111 Liver I 50 < 75 919 > 50 75 01 1 Spleen2) 5 50 I75 618 > 50 > 75 01 1 Kidney2) I50 < 75 818 > 50 575 01 1 I 50 I75 818 Ad rena 1 > 50 > 75 1/1 gland') Brain3) I 50 I75 616 > 50 > 75 11 1 The RNA samples were examined for the presence of ribosomal bands after size fractionation on the ethidiumstained gels, and were probed for the retinoblastoma (Rb) and p-actin transcripts. The extracted DNA was probed for the Rb gene. ') In two lung samples the DNA yield was insufficient for Southern blot analysis. )' The spleen, kidneys, and adrenal glands had been removed from one autopsy. )) Brain tissue was not available in three autopsies.
Lung')
500
r
RNA A N D DNA FROM AUTOPSIES
23.1 -
*
9.4 -
6.6-
4.4 -
2.3-
Fig. 2. Southern blot analysis of seven autopsy adrenal gland samples probed for the human retinoblastoma gene, DNA was digested with the restriction endonuclease EcoRI. Signals of varying intensity were obtained from all samples. HindII-digested hphage DNA was used as size marker. The postmortem sampling times are indicated.
DISCUSSION The RNA analyses of the autopsy and xenograft tissues were in good agreement, indicating that mRNA was appropriate for Northern blot analysis with the applied probes for approximately 50 h postmortem. Furthermore, the xenograft results suggested that this period may be extended if the tissue (corpse) is kept at lower temperatures.
1-
2.32.0
-
I ,
c-myc I
p-actin
Fig. 3. The agarose gel and corresponding Northern blot analysis of samples from a small cell lung cancer xenograft kept at 20°C from 0 to 72 h after excision. The blots were hybridized to probes for the c-ruf-1, c-myc, and p-actin transcripts. The molecular weight of transcripts was determined with reference to the 28s and 18s rRNA bands.
Histological examination showing only slight or no autolysis and the presence of ribosomal RNA after gel electrophoresis were indicative of the presence in the autopsy samples of mRNA appropriate for Northern blot analysis with the applied probes. The present investigations were carried out with probes recognizing gene transcripts of 2.1 to 4.7 kb and thus covering the size range of a large proportion of mRNAs. However, it should be emphasized that gene transcripts larger than that of the Rb gene (4.7 kb) may not be preserved to the same degree. Furthermore, the results showed only that autopsy material may be used for qualitative detection of gene transcripts. Partial degradation may have caused a decrease in the levels of expression as indicated by the different signal intensities obtained (Figs. 1 & 2). In other studies, mRNA was isolated from lung until 20 h (Hymphreys-Beher et al. 1986), from liver until 16 h (Finger et al. 1987), and from brain tissue until 36 h postmortem (Wood et al. 1986, Johnson et al. 1986, Morrison & GrijBn 1981). We detected RNA for longer periods in all tissues examined (Table 1). The xenograft experiment demonstrated that the RNA stability was strongly dependent on the temperature. The discrepancy between the present and other results might thus be ascribed to different conditions of postmortem tissue storage. As for RNA, the degree of DNA degradation was found to be dependent on the time lapse between death and autopsy (Table 1). However, DNA was generally preserved for longer periods, and the variation in stability among the organs examined was greater for DNA than for RNA. In all organs examined DNA was detected until three days postmortem, but in several cases DNA was isolated from tissues sampled five days postmortem. These findings are in fair agreement with the reported postmortem stability of 2 4 3 6 h in liver, five days in spleen, kidney and thyroid, and even longer in brain (Bur et al. 1988). In contrast to the RNA findings, DNA stability was not correlated with the histological grading of tissue autolysis. Several DNA samples from tissue graded as heavily autolysed provided a signal by Southern blot analysis. The results with respect to brain tissue repre50 1
LARSEN ef al.
sented a distinct divergence from the general pattern. Irrespective of the histological grading of the other organs, all brain samples showed only slight to moderate autolysis, and signals were obtained in almost all Northern and Southern blot analyses (Table l), independent of the postmortem sampling time. This is in concordance with the reported high frequency of isolation of undegraded DNA from autopsy brain tissue (Upadhyaya et al. 1985). In the xenograft investigation, there was a parallel variation in signal intensity with the three probes employed (Fig. 3). This was rather unexpected in view of the short in vivo half-life of the c-myc mRNA (10-20 min) (Dani et al. 1984) compared to the approximately 6-12 h reported for P-actin (Khalili& Weinmann 1984). One explanation for this extended c-myc mRNA stability could be a reduced cellular metabolic rate postmortem. Alternatively, the cells sustained unchanged mRNA production for two days following clinical death. This study was supported by the Weimann Foundation, the Michaelsen Foundation, and the Danish Cancer Society. The technical assistance of Jette R~hrmannis greatly appreciated.
REFERENCES Alitalo, K., Schwab, M . , Lin, C. C,,Varmus, H. E. & Bishop, J. M .:Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc. Natl. Acad. Sci. USA 80: 1707-171 I , 1983. Berendsen, H. H., de Leij, L., de Vries, E. G. E., Mesander, G., Mulder, N . H., de Jong, B., Buys, C. H. C.M., Postmus, I? E., Poppema, S., Sluiter, H. J. & The, H. T : Characterization of three small cell lung cancer cell lines established from one patient during longitudinal follow-up. Cancer Res. 48: 6891-6899, 1988. Bonner, T. I., Kerby, S. B., Sutrave, P,Gunnell, M . A., Mark, G. & Rapp, U. R. Structure and biological activity of human homologs of the raf/mil oncogene. Mol. Cell Biol. 5: 1400-1407, 1985. Bar, W , Kratzer, A., Machler, M. & Schmid, W: Postmortem stability of DNA. Forensic Sci. Int. 39: 59-70, 1988. Chomczynski, I! & Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-
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