140
KAI-OLAF NETZER
Total RNA (10-20 !!g)
Denaturing so1ution
2.0 !!1
6.0 !!1
Quickly chill on ice for 5 minutes. Spin briefly in a microfuge to collect
the sample. Keep the tube on ice.
Note: Ifthe desired amount ofRNA is present in a volume greater than 2 J..Ll,
the reaction can be scaled up. Always add 3 volumes of denaturing solution.
4. Layer the membrane on a wad of filter paper (e.g. Whatman 3MM)
soaked with 20x SSC.
5. Spot the RNA samples onto the filter in 4 J..Ll aliquots. Allow each spot to
dry between each application.
Note: The filter should remain damp but not too wet throughout the spotting of RNA samples.
6. Place the fllter membrane briefly on a sheet of dry filter paper (3MM) to
blot dry the membrane. Rinse briefly in 20x SSC.
7. Wrap the filter in plastic wrap while still damp. Fix RNA to the fllter by
irradiating the filter RNA side down on a UV transilluminator for 3-5
minutes. The filter is then ready for hybridization.
Note: For storage, nylon filters should be sealed in plastic bags and kept at
4°C.
Subprotocol 5
Preparation of Northern Blots
For N orthern blotting, RN A is first size-separated by denaturing agarose gel
electrophoresis, blotted to nitrocellulose or, more commonly, to a nylon
filter membrane, and hybridized. By analogy to Southern blotting which
involves blotting of DNA, this procedure has been named Northern blotting. Usually, Northern blots are used to detect transcription of specific
mRNA molecules in different cells, tissues, or organs, and to study regulation of transcription in response to various stimuli. Furthermore, N orthern
hybridization may be used to determine the length ofRNA transcripts. Alternative splicing, for example, may be detected by variable transcript
lengths. Densitometry and comparison with controls permits quantification of RNA molecules in order to characterize the level of expression.
KAI-OLAF NETZER
Total RNA (10-20 !!g)
Denaturing so1ution
2.0 !!1
6.0 !!1
Quickly chill on ice for 5 minutes. Spin briefly in a microfuge to collect
the sample. Keep the tube on ice.
Note: Ifthe desired amount ofRNA is present in a volume greater than 2 J..Ll,
the reaction can be scaled up. Always add 3 volumes of denaturing solution.
4. Layer the membrane on a wad of filter paper (e.g. Whatman 3MM)
soaked with 20x SSC.
5. Spot the RNA samples onto the filter in 4 J..Ll aliquots. Allow each spot to
dry between each application.
Note: The filter should remain damp but not too wet throughout the spotting of RNA samples.
6. Place the fllter membrane briefly on a sheet of dry filter paper (3MM) to
blot dry the membrane. Rinse briefly in 20x SSC.
7. Wrap the filter in plastic wrap while still damp. Fix RNA to the fllter by
irradiating the filter RNA side down on a UV transilluminator for 3-5
minutes. The filter is then ready for hybridization.
Note: For storage, nylon filters should be sealed in plastic bags and kept at
4°C.
Subprotocol 5
Preparation of Northern Blots
For N orthern blotting, RN A is first size-separated by denaturing agarose gel
electrophoresis, blotted to nitrocellulose or, more commonly, to a nylon
filter membrane, and hybridized. By analogy to Southern blotting which
involves blotting of DNA, this procedure has been named Northern blotting. Usually, Northern blots are used to detect transcription of specific
mRNA molecules in different cells, tissues, or organs, and to study regulation of transcription in response to various stimuli. Furthermore, N orthern
hybridization may be used to determine the length ofRNA transcripts. Alternative splicing, for example, may be detected by variable transcript
lengths. Densitometry and comparison with controls permits quantification of RNA molecules in order to characterize the level of expression.