Analysis of RNA by Northern and Slot‐Blot Hybridization

互联网2013-12-31

1067

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Specific sequences in RNA preparations can be detected by blotting and hybridization analysis using techniques very similar to those originally developed for DNA. Fractionated RNA is transferred from an agarose gel to a membrane support (northern blotting), while unfractionated RNA is immobilized by slot or dot blotting. The resulting blots are studied by hybridization analysis with labeled DNA or RNA probes. Included in this unit are detailed procedures for RNA denaturation, blotting and hybridization. Also described is a method for stripped hybridization probes from blots so the blots can be re?hybridized with a different probe.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose‐Formaldehyde Gel Electrophoresis
Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment
Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting
Support Protocol 1: Removal of Probes from Northern Blots
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose‐Formaldehyde Gel Electrophoresis

Materials

10× and 1× MOPS running buffer (see recipe for 10× buffer)
12.3 M (37%) formaldehyde, pH >4.0
RNA sample: total cellular RNA ( appendix 1I ) or poly(A)⁺ RNA ( appendix 1I )
Formamide
Formaldehyde loading buffer (see recipe )
0.5 M ammonium acetate and 0.5 µg/ml ethidium bromide in 0.5 M ammonium acetate or 10 mM sodium phosphate (pH 7.0; see recipe )/1.1 M formaldehyde with and without 10 µg/ml acridine orange
0.24‐ to 9.5‐kb RNA ladder (Life Technologies) (optional)
0.05 M NaOH/1.5 M NaCl (optional)
0.5 M Tris⋅Cl (pH 7.4; appendix 2A )/1.5 M NaCl (optional)
20×, 2×, and 6× SSC ( appendix 2A )
0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2 (optional)

DNA suitable for use as probe (unit 5.1 ) or for in vitro transcription to make RNA probe (Table 5.17.1 )

Table 5.7.1 MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases Properties of Materials Used for Immobilization of Nucleic Acids b Properties of Materials Used for Immobilization of Nucleic Acids

Vector	Size (bp)	Markers a	Promoters
pBluescript	2950	amp, lacZ ′	T3, T7
pGEM series	2746‐3223	amp, lacZ ′	SP6, T7
pGEMEX‐1	4200	amp	SP6, T3, T7
pSELECT‐1	3422	tet, lacZ ′	SP6, T7
pSP18, 19, 64, 65	2999‐3010	amp	SP6
pSP70, 71, 72, 73	2417‐2464	amp	SP6, T7
pSPORT1	4109	amp, lacZ ′	SP6, T7
pT3/T7 series	2700, 2950	amp, lacZ ′	T3, T7
pWE15	8800	amp, neo	T3, T7
pWE16	8800	amp, dhfr	T3, T7
	Nitrocellulose	Supported nitrocellulose	Uncharged nylon	Positively charged nylon	Activated papers
Application	ssDNA, RNA, protein	ssDNA, RNA, protein	ssDNA, dsDNA, DNA, protein	ssDNA, dsDNA, RNA, protein	ssDNA, RNA
Binding capacity (µg nucleic acid/cm² )	80‐100	80‐100	400‐600	400‐600	2‐40
Tensile strength	Poor	Good	Good	Good	Good
Mode of nucleic acid attachment c	Noncovalent	Noncovalent	Covalent	Covalent	Covalent
Lower size limit for efficient nucleic acid retention	500 nt	500 nt	50 nt or bp	50 nt or bp	5 nt
Suitability for reprobing	Poor (fragile)	Poor (loss of signal)	Good	Good	Good
Commercial examples	Schleicher & Schuell BA83, BA85;Amersham Hybond‐C	Schleicher & Schuell Optibond;Amersham Hybond‐C extra	Amersham Hybond‐N;Stratagene Duralon‐UV;Du Pont NEN GeneScreen	Schleicher & Schuell Nytran;Amersham Hybond‐N⁺ ;Bio‐Rad ZetaProbe;PALL Biodyne B;Du Pont NEN GeneScreen Plus	Schleicher & Schuell APT papers

^a Abbreviations: amp, ampicillin resistance; dhfr, dihydrofolate reductase; lacZ , β‐galactosidase α‐peptide; neo, neomycin phosphotransferase (kanamycin resistance); tet, tetracycline resistance.

Formamide prehybridization/hybridization solution (see recipe )
2× SSC/0.1% (w/v) SDS
0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
0.1× SSC/0.1% (w/v) SDS, 68°C

55°, 60°, and 100°C water baths
Oblong sponge slightly larger than the gel being blotted
RNase‐free glass dishes (baked for 4 hr at 300°C)
Whatman 3MM filter paper sheets
UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)

Nitrocellulose or nylon membrane (see Table 5.17.2 for list of suppliers)

Table 5.7.2 MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases Properties of Materials Used for Immobilization of Nucleic Acids b Properties of Materials Used for Immobilization of Nucleic Acids

Vector	Size (bp)	Markers a	Promoters
pBluescript	2950	amp, lacZ ′	T3, T7
pGEM series	2746‐3223	amp, lacZ ′	SP6, T7
pGEMEX‐1	4200	amp	SP6, T3, T7
pSELECT‐1	3422	tet, lacZ ′	SP6, T7
pSP18, 19, 64, 65	2999‐3010	amp	SP6
pSP70, 71, 72, 73	2417‐2464	amp	SP6, T7
pSPORT1	4109	amp, lacZ ′	SP6, T7
pT3/T7 series	2700, 2950	amp, lacZ ′	T3, T7
pWE15	8800	amp, neo	T3, T7
pWE16	8800	amp, dhfr	T3, T7
	Nitrocellulose	Supported nitrocellulose	Uncharged nylon	Positively charged nylon	Activated papers
Application	ssDNA, RNA, protein	ssDNA, RNA, protein	ssDNA, dsDNA, DNA, protein	ssDNA, dsDNA, RNA, protein	ssDNA, RNA
Binding capacity (µg nucleic acid/cm² )	80‐100	80‐100	400‐600	400‐600	2‐40
Tensile strength	Poor	Good	Good	Good	Good
Mode of nucleic acid attachment c	Noncovalent	Noncovalent	Covalent	Covalent	Covalent
Lower size limit for efficient nucleic acid retention	500 nt	500 nt	50 nt or bp	50 nt or bp	5 nt
Suitability for reprobing	Poor (fragile)	Poor (loss of signal)	Good	Good	Good
Commercial examples	Schleicher & Schuell BA83, BA85;Amersham Hybond‐C	Schleicher & Schuell Optibond;Amersham Hybond‐C extra	Amersham Hybond‐N;Stratagene Duralon‐UV;Du Pont NEN GeneScreen	Schleicher & Schuell Nytran;Amersham Hybond‐N⁺ ;Bio‐Rad ZetaProbe;PALL Biodyne B;Du Pont NEN GeneScreen Plus	Schleicher & Schuell APT papers

^b This table is based on Brown ( ), with permission from BIOS Scientific Publishers Ltd.

^c After suitbale immobilization procedure (see text).

Glass plate slightly larger than final membrane size
Vacuum oven
UV transilluminator, calibrated
Hybridization oven (e.g., Hybridiser HB‐1, Techne) and tubes
Additional reagents and equipment for agarose gel electrophoresis ( appendix 1N ), radiolabeling of DNA by nick translation or random oligonucleotide priming (unit 4.2 ), RNA labeling by in vitro synthesis (unit 4.3 ), measuring specific activity of labeled nucleic acids and separating unincorporated nucleotides from labeled nucleic acids (CPMB UNIT and appendix 1A in this manual), and autoradiography (CPMB APPENDIX and appendix 1A in this manual)

NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in appendix 2A ; see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.

Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment

10 mM and 100 mM sodium phosphate, pH 7.0 (see recipe )
Dimethyl sulfoxide (DMSO)
6 M (40%) glyoxal, deionized immediately before use (see recipe )
Glyoxal loading buffer (see recipe )
20 mM Tris⋅Cl, pH 8.0 ( appendix 2A )
Apparatus for recirculating running buffer during electrophoresis
50° and 65°C water baths

NOTE: All solutions should be prepared with sterile deionized water that has been treated with DEPC as described in appendix 2A see unit introduction for further instructions and precautions regarding establishment of an RNase‐free environment.

Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting

0.1 M NaOH
10× SSC ( appendix 2A )
20× SSC , room temperature and ice‐cold
Denaturing solution (see recipe )
100 mM sodium phosphate, pH 7.0 (see recipe )
Dimethyl sulfoxide (DMSO)
6 M (40%) glyoxal, deionized immediately before use (see recipe )
Manifold apparatus with a filtration template for slot blots (e.g., Bio‐Rad Bio‐Dot SF, Schleicher and Schuell Minifold II)
50° and 60°C water baths

Support Protocol 1: Removal of Probes from Northern Blots

Materials

Northern hybridization membrane containing probe (see protocol 1 , protocol 2 , or protocol 3 )
Stripping solution (see recipe )
Hybridization bags
65°, 80°, or 100° (boiling) water bath
UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)

Additional reagents and equipment for autoradiography (see CPMB APPENDIX and appendix 1A in this manual)

CAUTION: If hybridization probes include a radioactive label, dispose of stripping solutions as radioactive waste. Observe appropriate caution when working with the toxic compound formamide.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 5.17.1 Rat liver RNA (5 µg) was electrophoresed on a formaldehyde 1% agarose gel containing ethidium bromide (left), transferred to a hybridization membrane and stained with methylene blue stain (Molecular Research Center; Herrin and Schmidt, , right). Shown are 28S (4.7 kb) and 18S (1.9 kb) ribosomal RNAs, as well as 4S to 5S (0.10 to 0.15 kb) RNA containing mix of tRNA and 5S ribosomal RNA.

View Image

Videos

Literature Cited

Literature Cited
	Alwine, J.C., Kemp, D.J., and Stark, G.R. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl‐paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74:5350‐5354.
	Bailey, J.M. and Davidson, N. 1976. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal. Biochem. 70:75‐85.
	Bodkin, D.K. and Knudson, D.L. 1985. Assessment of sequence relatedness of double‐stranded RNA genes by RNA‐RNA blot hybridization. J. Virol. Methods 10:45‐52.
	Brown, T.A. (ed.) 1991. Molecular Biology Labfax. BIOS Scientific Publishers, Oxford.
	Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucl. Acids Res. 4:1539‐1552.
	Chomczynski, P. 1992. One‐hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201:134‐139.
	Herrin, D.L. and Schmidt, G.W. 1988. Rapid, reversible staining of Northern blots prior to hybridization. BioTechniques 6:196‐200.
	Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Res. 7:1541‐1552.
	Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions: A critical reexamination. Biochemistry 16:4743‐4751.
	Peferoen, M., Huybrechts, R., and De Loof, A. 1982. Vacuum‐blotting: A new simple and efficient transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to nitrocellulose. FEBS Lett. 145:369‐372.
	Smith, M.R., Devine, C.S., Cohn, S.M., and Lieberman, M.W. 1984. Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose. Anal. Biochem. 137:120‐124.
	Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
	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.
	Wilkinson, M. 2000. Purification of RNA. In Essential Molecular Biology:A Practical Approach 2nd edition., Vol. 1 (T.A. Brown, ed.) pp. 69‐88. Oxford University Press, Oxford.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Analysis of RNA by Northern and Slot‐Blot Hybridization

Abstract

Table of Contents

Materials

Basic Protocol 1: Northern Hybridization of RNA Fractionated by Agarose‐Formaldehyde Gel Electrophoresis

Alternate Protocol 1: Northern Hybridization of RNA Denatured by Glyoxal/DMSO Treatment

Alternate Protocol 2: Northern Hybridization of Unfractionated RNA Immobilized by Slot Blotting

Support Protocol 1: Removal of Probes from Northern Blots

Figures

Videos

Literature Cited