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Isolation and Northern blotting of RNA from Aspergillus nidulans
Journal of Microbiological Methods 15 (1992) 1 - 5 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167- 7012/92/$ 5.00 MIMET 00466
Isolation and Northern blotting of RNA from
Aspergillus nidulans R.E. Bradshaw and T.M. Pillar Leicester Biocentre, University of Leicester, Leicester, UK (Received 12 August 1991; accepted 27 September 1991)
Summary We present a reliable method for the extraction and Northern blotting of RNA from the filamentous fungus Aspergillus nidulans. The use of glass beads to grind open the fungal mycelium means that samples can be processed very quickly for RNA isolation. A combination of glyoxal/dimethylsulfoxide (Me_,SO) RNA d,:~aturation with a thin horizontal agarose/MOPS gel gives consistently high-quality Northern blot.s,
Key words: Aspergillus nidulans; Northern blotting; RNA isolation
!ntroducti,~n Current popular methods for RNA isolation from filamentous fungi include the grinding of lyophilized mycelium in the presence of liquid N 2 [I] and the use of guanidinium thiocyanate as a chaotrophic agent [2]. The initial stages of grinding mycelium can be slow and cumbersome, particularly if many different samples are required; for example, in studies of transcriptional ;egulation. Northern blotting usually involves handling gels containing formaldehyde or methyl mercuric hydroxide [3], both of which are extremely toxic. We present here a combination of an RNA extraction method, using glass beads to disrupt Aspergillus nidulans mycelium, with a glyoxal/dimethylsulfoxide (Me2SO) Northern blotting technique adapted from Thomas [4]. These methods allow the rapid isolation of high-quality RNA from different mycelium samples and the reliable generation of Northern blots. The total RNA obtained includes mRNA suitable for reverse transcription, as confirmed by primer extension analysis. Correspondence to: R. E. Bradshaw, Leicester Biocentre, University of Leicester, Leicester LEI 7ND, UK.
Materials and Methods
Materials Sorbitol lysis buffer: 700 mM sorbitol, 50 mM Tris[hydroxylmethyl]aminomethane (Tris) pH 7.5, 10 mM ethylene diamine tetra-acetic acid (EDTA). DNase buffer: 100 mM sodium acetate (NaOAc), 5 mM MgSO4;pH 5. MOPS buffer (20 ×): 400 mM 3-[N-morpholino] propane-sulfonic acid (MOPS), 60 mM NaOAc, 20 mM EDTA; pH 7. SSC (20 x): 3 M NaCI, 300 mM sodium citrate; pH 7. SSPE (20×): 3 M NaCI, 175 mM NaH2PO4.2H20, 20 mM EDTA; pH 7.4. TE: 10 mM Tris pH 7.5, 1 mM EDTA. Me2SO: dimethylsulfoxide (Fluka, analytical grade, 41640). Glyoxal: (Sigma, G-3140: 40°7o aqueous solution), deionized on mixed-bed resin (Bio-Rad, AG 501-X8) and stored in aliquots at - 8 0 ° C under autoclaved light mineral oil (Sigma, M-3516). Glass beads: (B. Braun, Melsungen AG, product no. 854 170/1), diameter 0.45 - 0.50 mm, acid washed in 1 M HCI overnight, rinsed thoroughly to pH 7, dried and baked at 1 5 0 ° C > 4 h. Used beads are recycled in same way. Glassware was baked at 150°C for at least 4 h to inactivate ribonucleases and, where appropriate, solutions autoclaved at 15 psi, 121 °C for 20 min.
Growth of mycefium Conidiospores of a haploid strain of A. nidulans (R153: pyroA4, wA3) were inoculated (106.ml - l) into siliconized flasks containing Aspergiilus minimal medium [5] supplemented with 0.5°-/o yeast extract (Difco) and 0.2°7o casamino acids (Difco). Cultures were grown at 30°C, 180 rpm for 20 h, then chilled on ice for 5 - 10 min prior to harvesting by vacuum filtration using sterile filter papers.
RNA extraction Mycelium was washed with ice-cold water (vacuum filtration) and quickly transferred to a 30-ml siliconized glass Corex tube (Du Pont, no. 00156) containing ice-cold sorbitol lysis buffer (1.5 g wet weight of mycelium to 1.5 ml buffer). Then, 50 #1 /3-mercaptoethanoi (Sigma, M-6250) and cold glass beads ( ~ 3 g weight) were added: the mixture was vortexed for 60 s prior to rapid freezing on dry ice. At this stage it is convenient to store the frozen samples at - 8 0 ° C (indefinitely if desired). The following were added to the frozen pellet: 2.5 ml H,O, 300 #I 1 M Tris (pH 8) and 500 ~1 100-/0 sodium dodecyl sulphate (SDS). The tube and its frozen contents were placed in a boiling water bath until thawed (2--3 min) and were subsequently vortexed for 20 s. Several phenol/chloroform/isoamylalcohol [IAA] (25/24/1)extractions were performed (until the interface was clear), followed by a final chloroform/IAA (24/1) extraction. Nucleic acids were precipitated with ethanol + 0.3 M NHaAc for 5 - 10 min on ice (at this stage there is a tendency for excess salts to be precipitated along with the nucleic acids below this temperature). Following resuspension in TE the nucleic acids were quantitated by 00260absorption.
RNA gel and blotting DNase treatment of a 20-#g RNA sample was carried out usi~g J LI of RNase-free DNase (Mannheim Boehringer, 776 785) in 200 tLl DNase buffer at 37 °C for 15 min. RNA was then re-extracted with phenol/chloroform/IAA, ethanol-precipitated (0.3 M NHaAc), the pellet dried in a 37 °C incubator for 5 mia and resuspended in 4 #1 H20. A mixture of deionized glyoxal, 20 x MOPS buffer and Me2SO was prepared in a separate Eppendorf tube in the proportions 50:8:100 and 16 #1 of the mixture was added to each 4/~I of resuspended RNA (to give final concentrations of 10070 giyoxai, i × MOPS, 50°7/0 Me2SO v/v). After thorough vortexing, the RNA suspension was incubated, under a layer of light mineral oil, at 50°C for 60 min. A thin gel was poured on a flat 12.5 x 10-cm glass plate. Using 25 ml of 1070agarose (Miles HSB) in 1 x MOPS buffer, and relying on surface tension, a gel thickness of 2 mm was achieved. We loaded up to 12 samples, each with 7 #g total RNA (i.e., 7 #1 from the 20-#1 reaction mix), on a gel this size. RNA samples were loaded directly from the glyoxalation reactions and a tracker dye was included in one lane: the Me2SO/glyoxal mix, being more dense than 1 × MOPS buffer, pipettes easily into the gel pockets. The gel was run at 70 V for 3 h at 4 °C: buffer circulatiov, was unnecessary. Gels were stained with ethidium bromide (0.25 # g - m l - t ) and photographed after running the gel. Without further treatment the RNA was transferred to a nylon membrane (Amersham, Hybond N) by capillary blotting with 20 × SSC (overnight at 4 °C), making sure that the underneath of the gel (as poured) was in contact with the nylon membrane. RNA was fixed onto the unwashed membrane by UV crosslinking and the glyoxalation was reversed by baking at 80 °C for 2 h in a vacuum oven. Northern hybridization Northern blots were prehybridized for at least 4 h in buffer (5 x SSPE, 5 × Denhardt's solution, 0.2°70 SDS, 50 #g/ml sonicated salmon sperm DNA). Fragments of Dr~T^ . . . . . . . . . ,4;..,. . .l l A . ,,,uu,un~ -:'J'" [i f - 32p]dCTP ~ c ~ w ~ labelled --'*'a.,It,a ~.,ILyll~..,,.~l,,tl,../llg.glll ~ ,.. tO ! I .X.I I.I., ..) W WI[II using hexamer priming [6] and used to probe the Northern blots at 65 °C for 2 - 3 days. Filters were washed at room temperature with 3 × SSC, 0.2070 SDS. Primer extension Total RNA was precipitated with an equal volume of 6 M lithium acetate pH 6 (in order to remove DNA and polysaccharides) and primer extension was carried out using a synthetic oligonucleotide primer complementary to a known mRNA species, essentially as described by Praekelt and Meacock [7]. Results and Discussion
Although the fragmentation of mycelium by vortexing with glass beads was by no means complete, yields of nucleic acids were substantial: = 1 mg total RNA from 1 g wet weight of mycelium. By meticulous care in the preparation and use of solutions it was not necessary to use RNase inhibitors such as diethylpyrocarbonate (DEPC) in order to obtain RNA of a consistently pure quality. The use of glass beads and the freeze-thaw method to break open mycelium facilitates the handling of separate RNA extractions and the coordination cf samples harvested at different times.
The use of a thin surface tension gel lead to an efficient transfer of glyo×alated RNA onto nylon membrane and sharp discrete mRNA signals following hybridization with homologous DNA probes (Fig. 1, panel I). Both nuclearly encoded (cytochrome c) and mitochondrially encoded (cytochrome b) mRNA transcripts were detected. A DNA probe prepared from the putative ribosomal protein S16 gene hybridised to two transcript species: the sizes of these transcripts are consistent with the expected sizes of the unspliced and mature mRNA, based on sequence analysis (manuscript in preparation). Thus this RNA extraction method allows the isolation of intron-containing mRNA precursors as well as mature transcripts. Primer extension reactions were carried out using total RNA and an end-labelled oligonucleotide complementary to part of the ribosomal protein S16 gene coding sequence. Clear discrete extension products were obtained (Fig. 1, panel II), indicating that the quality of the mRNA is suitable for reverse transcription. The methods outlined here for RNA extraction and Northern blotting are currently used in our laboratory for the yeast Saccharomyces cerevisiae as well as for A. nidulans, hence are applicable to a wide range of fungal species.
I CYC
COB
1.7-
RP
m
1.10.7-
~
kb
P.
Fig. 1.
G
A
Panel 1: Northern blot of total cellular RNA (7 ug) probed with radiolabelled DNA fragments of
A. nidulans genes as indicated. Lanes: CYC, cytochrome c; COB, cytochrome b; RP, ribosomal protein
S16. Panel 11: primer extension from 50 ug total RNA (annealed with a 28-base oligonucleotide homologous to ribosomal protein S16 gene) and run on a 6°70 polyacrylamide denaturing gel: otie-third of reaction products were loaded (lane P) alongside an appropriate dideoxy sequencing ladder (ddGTP and ddATP shown; lanes G and A). Arrow indicates major primer extension product.
References 1 Reinert, W. R., Patel, V. B. and Giles, N. H. (1981) Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency on qa-! function. Mol. Cell. Biol. 1,829- 835. 2 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuc!ease. Biochemistry !8, 5294- 5299. 3 Maniatis, T., Fritsch. E. F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 4 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 77, 5201 -5205. 5 Pontecorvo, G., Roper, J.A., Hemmons, D.W., MacDonald, K.D. and Bufton, A.W. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141 - 238. 6 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 - 13. 7 Praekelt, U.M. and Meacock, P.A. (1990) HSPI2, a new small heat shock ge,ae of Saccharomyces cerevisiae. Mol. Gen. Genet. 223, 9 7 - 106.
Isolation and Northern blotting of RNA from
Aspergillus nidulans R.E. Bradshaw and T.M. Pillar Leicester Biocentre, University of Leicester, Leicester, UK (Received 12 August 1991; accepted 27 September 1991)
Summary We present a reliable method for the extraction and Northern blotting of RNA from the filamentous fungus Aspergillus nidulans. The use of glass beads to grind open the fungal mycelium means that samples can be processed very quickly for RNA isolation. A combination of glyoxal/dimethylsulfoxide (Me_,SO) RNA d,:~aturation with a thin horizontal agarose/MOPS gel gives consistently high-quality Northern blot.s,
Key words: Aspergillus nidulans; Northern blotting; RNA isolation
!ntroducti,~n Current popular methods for RNA isolation from filamentous fungi include the grinding of lyophilized mycelium in the presence of liquid N 2 [I] and the use of guanidinium thiocyanate as a chaotrophic agent [2]. The initial stages of grinding mycelium can be slow and cumbersome, particularly if many different samples are required; for example, in studies of transcriptional ;egulation. Northern blotting usually involves handling gels containing formaldehyde or methyl mercuric hydroxide [3], both of which are extremely toxic. We present here a combination of an RNA extraction method, using glass beads to disrupt Aspergillus nidulans mycelium, with a glyoxal/dimethylsulfoxide (Me2SO) Northern blotting technique adapted from Thomas [4]. These methods allow the rapid isolation of high-quality RNA from different mycelium samples and the reliable generation of Northern blots. The total RNA obtained includes mRNA suitable for reverse transcription, as confirmed by primer extension analysis. Correspondence to: R. E. Bradshaw, Leicester Biocentre, University of Leicester, Leicester LEI 7ND, UK.
Materials and Methods
Materials Sorbitol lysis buffer: 700 mM sorbitol, 50 mM Tris[hydroxylmethyl]aminomethane (Tris) pH 7.5, 10 mM ethylene diamine tetra-acetic acid (EDTA). DNase buffer: 100 mM sodium acetate (NaOAc), 5 mM MgSO4;pH 5. MOPS buffer (20 ×): 400 mM 3-[N-morpholino] propane-sulfonic acid (MOPS), 60 mM NaOAc, 20 mM EDTA; pH 7. SSC (20 x): 3 M NaCI, 300 mM sodium citrate; pH 7. SSPE (20×): 3 M NaCI, 175 mM NaH2PO4.2H20, 20 mM EDTA; pH 7.4. TE: 10 mM Tris pH 7.5, 1 mM EDTA. Me2SO: dimethylsulfoxide (Fluka, analytical grade, 41640). Glyoxal: (Sigma, G-3140: 40°7o aqueous solution), deionized on mixed-bed resin (Bio-Rad, AG 501-X8) and stored in aliquots at - 8 0 ° C under autoclaved light mineral oil (Sigma, M-3516). Glass beads: (B. Braun, Melsungen AG, product no. 854 170/1), diameter 0.45 - 0.50 mm, acid washed in 1 M HCI overnight, rinsed thoroughly to pH 7, dried and baked at 1 5 0 ° C > 4 h. Used beads are recycled in same way. Glassware was baked at 150°C for at least 4 h to inactivate ribonucleases and, where appropriate, solutions autoclaved at 15 psi, 121 °C for 20 min.
Growth of mycefium Conidiospores of a haploid strain of A. nidulans (R153: pyroA4, wA3) were inoculated (106.ml - l) into siliconized flasks containing Aspergiilus minimal medium [5] supplemented with 0.5°-/o yeast extract (Difco) and 0.2°7o casamino acids (Difco). Cultures were grown at 30°C, 180 rpm for 20 h, then chilled on ice for 5 - 10 min prior to harvesting by vacuum filtration using sterile filter papers.
RNA extraction Mycelium was washed with ice-cold water (vacuum filtration) and quickly transferred to a 30-ml siliconized glass Corex tube (Du Pont, no. 00156) containing ice-cold sorbitol lysis buffer (1.5 g wet weight of mycelium to 1.5 ml buffer). Then, 50 #1 /3-mercaptoethanoi (Sigma, M-6250) and cold glass beads ( ~ 3 g weight) were added: the mixture was vortexed for 60 s prior to rapid freezing on dry ice. At this stage it is convenient to store the frozen samples at - 8 0 ° C (indefinitely if desired). The following were added to the frozen pellet: 2.5 ml H,O, 300 #I 1 M Tris (pH 8) and 500 ~1 100-/0 sodium dodecyl sulphate (SDS). The tube and its frozen contents were placed in a boiling water bath until thawed (2--3 min) and were subsequently vortexed for 20 s. Several phenol/chloroform/isoamylalcohol [IAA] (25/24/1)extractions were performed (until the interface was clear), followed by a final chloroform/IAA (24/1) extraction. Nucleic acids were precipitated with ethanol + 0.3 M NHaAc for 5 - 10 min on ice (at this stage there is a tendency for excess salts to be precipitated along with the nucleic acids below this temperature). Following resuspension in TE the nucleic acids were quantitated by 00260absorption.
RNA gel and blotting DNase treatment of a 20-#g RNA sample was carried out usi~g J LI of RNase-free DNase (Mannheim Boehringer, 776 785) in 200 tLl DNase buffer at 37 °C for 15 min. RNA was then re-extracted with phenol/chloroform/IAA, ethanol-precipitated (0.3 M NHaAc), the pellet dried in a 37 °C incubator for 5 mia and resuspended in 4 #1 H20. A mixture of deionized glyoxal, 20 x MOPS buffer and Me2SO was prepared in a separate Eppendorf tube in the proportions 50:8:100 and 16 #1 of the mixture was added to each 4/~I of resuspended RNA (to give final concentrations of 10070 giyoxai, i × MOPS, 50°7/0 Me2SO v/v). After thorough vortexing, the RNA suspension was incubated, under a layer of light mineral oil, at 50°C for 60 min. A thin gel was poured on a flat 12.5 x 10-cm glass plate. Using 25 ml of 1070agarose (Miles HSB) in 1 x MOPS buffer, and relying on surface tension, a gel thickness of 2 mm was achieved. We loaded up to 12 samples, each with 7 #g total RNA (i.e., 7 #1 from the 20-#1 reaction mix), on a gel this size. RNA samples were loaded directly from the glyoxalation reactions and a tracker dye was included in one lane: the Me2SO/glyoxal mix, being more dense than 1 × MOPS buffer, pipettes easily into the gel pockets. The gel was run at 70 V for 3 h at 4 °C: buffer circulatiov, was unnecessary. Gels were stained with ethidium bromide (0.25 # g - m l - t ) and photographed after running the gel. Without further treatment the RNA was transferred to a nylon membrane (Amersham, Hybond N) by capillary blotting with 20 × SSC (overnight at 4 °C), making sure that the underneath of the gel (as poured) was in contact with the nylon membrane. RNA was fixed onto the unwashed membrane by UV crosslinking and the glyoxalation was reversed by baking at 80 °C for 2 h in a vacuum oven. Northern hybridization Northern blots were prehybridized for at least 4 h in buffer (5 x SSPE, 5 × Denhardt's solution, 0.2°70 SDS, 50 #g/ml sonicated salmon sperm DNA). Fragments of Dr~T^ . . . . . . . . . ,4;..,. . .l l A . ,,,uu,un~ -:'J'" [i f - 32p]dCTP ~ c ~ w ~ labelled --'*'a.,It,a ~.,ILyll~..,,.~l,,tl,../llg.glll ~ ,.. tO ! I .X.I I.I., ..) W WI[II using hexamer priming [6] and used to probe the Northern blots at 65 °C for 2 - 3 days. Filters were washed at room temperature with 3 × SSC, 0.2070 SDS. Primer extension Total RNA was precipitated with an equal volume of 6 M lithium acetate pH 6 (in order to remove DNA and polysaccharides) and primer extension was carried out using a synthetic oligonucleotide primer complementary to a known mRNA species, essentially as described by Praekelt and Meacock [7]. Results and Discussion
Although the fragmentation of mycelium by vortexing with glass beads was by no means complete, yields of nucleic acids were substantial: = 1 mg total RNA from 1 g wet weight of mycelium. By meticulous care in the preparation and use of solutions it was not necessary to use RNase inhibitors such as diethylpyrocarbonate (DEPC) in order to obtain RNA of a consistently pure quality. The use of glass beads and the freeze-thaw method to break open mycelium facilitates the handling of separate RNA extractions and the coordination cf samples harvested at different times.
The use of a thin surface tension gel lead to an efficient transfer of glyo×alated RNA onto nylon membrane and sharp discrete mRNA signals following hybridization with homologous DNA probes (Fig. 1, panel I). Both nuclearly encoded (cytochrome c) and mitochondrially encoded (cytochrome b) mRNA transcripts were detected. A DNA probe prepared from the putative ribosomal protein S16 gene hybridised to two transcript species: the sizes of these transcripts are consistent with the expected sizes of the unspliced and mature mRNA, based on sequence analysis (manuscript in preparation). Thus this RNA extraction method allows the isolation of intron-containing mRNA precursors as well as mature transcripts. Primer extension reactions were carried out using total RNA and an end-labelled oligonucleotide complementary to part of the ribosomal protein S16 gene coding sequence. Clear discrete extension products were obtained (Fig. 1, panel II), indicating that the quality of the mRNA is suitable for reverse transcription. The methods outlined here for RNA extraction and Northern blotting are currently used in our laboratory for the yeast Saccharomyces cerevisiae as well as for A. nidulans, hence are applicable to a wide range of fungal species.
I CYC
COB
1.7-
RP
m
1.10.7-
~
kb
P.
Fig. 1.
G
A
Panel 1: Northern blot of total cellular RNA (7 ug) probed with radiolabelled DNA fragments of
A. nidulans genes as indicated. Lanes: CYC, cytochrome c; COB, cytochrome b; RP, ribosomal protein
S16. Panel 11: primer extension from 50 ug total RNA (annealed with a 28-base oligonucleotide homologous to ribosomal protein S16 gene) and run on a 6°70 polyacrylamide denaturing gel: otie-third of reaction products were loaded (lane P) alongside an appropriate dideoxy sequencing ladder (ddGTP and ddATP shown; lanes G and A). Arrow indicates major primer extension product.
References 1 Reinert, W. R., Patel, V. B. and Giles, N. H. (1981) Genetic regulation of the qa gene cluster of Neurospora crassa: induction of qa messenger ribonucleic acid and dependency on qa-! function. Mol. Cell. Biol. 1,829- 835. 2 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuc!ease. Biochemistry !8, 5294- 5299. 3 Maniatis, T., Fritsch. E. F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 4 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. 77, 5201 -5205. 5 Pontecorvo, G., Roper, J.A., Hemmons, D.W., MacDonald, K.D. and Bufton, A.W. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141 - 238. 6 Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6 - 13. 7 Praekelt, U.M. and Meacock, P.A. (1990) HSPI2, a new small heat shock ge,ae of Saccharomyces cerevisiae. Mol. Gen. Genet. 223, 9 7 - 106.