Protocol
An Alternative Method for Processing Northern Blots After Capillary Transfer Timothy W. Nilsen
Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Bovine serum albumin (BSA) EDTA (0.5 M, pH 8.0) Formamide Northern blot (i.e., a membrane containing RNA fragments that have been transferred from a gel by capillary transfer) The procedure described below is performed after capillary transfer (for capillary transfer, see Steps 1–5 in Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions [Rio 2015]).
Probe (radiolabeled, specific to the RNA of interest) SDS (20%, w/v) Sodium phosphate buffer for Church and Gilbert hybridization SSC buffer for northerns (20×) The 20× stock must be diluted for use in Step 4.
Adapted from RNA: A Laboratory Manual by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
314
Alternative Method for Processing Northern Blots
Equipment
Aluminum foil Geiger counter (optional; see Step 31) Incubator with rotating or rocking platform (42˚C) Kimwipes (optional; see Step 29) Oven (80˚C) Paper towels Pencil Plastic container with lid to hold and mix 500 mL of liquid Plastic wrap Platform shaker at room temperature Rinse container with lid Scalpel or razor blade Scissors Styrofoam and lead pigs (optional; see Step 27) UV cross-linker with 254-nm bulbs (e.g., Stratalinker, Stratagene) Alternatively, use a 254-nm UV transilluminator (see Step 6).
Vacuum food sealer and bags Water bath (shaking) at temperature appropriate for desired stringency (see Step 22) X-ray film and developer (or phosphorimaging equipment)
METHOD Cross-Linking Perform Steps 1–5 quickly and carefully to prevent the agarose from drying and sticking to the membrane, causing unnecessary background. Do not allow the membrane to dry out at any stage during the procedure.
1. On the day after performing the capillary transfer procedure (see Steps 1–5 in Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions [Rio 2015]), remove the paper towels and the blot papers from the transfer setup. 2. Mark the locations of the wells on the membrane with a pencil. Orient the gel and the membrane by slicing off or notching a corner, using a scalpel or razor blade. 3. Peel the gel off of the membrane and discard it. 4. Rinse the membrane in 2× SSC to remove any agarose that may remain stuck to it. Gently rub the membrane with a gloved hand to be sure that the agarose is removed. 5. Place the membrane on a foil sheet with the RNA surface facing up. 6. Cross-link the RNA to the membrane by exposing the membrane to short-wave UV light at 254 nm (use the 1200-mJ auto-cross-link setting in a Stratalinker or place the membrane facedown on a 254-nm light box for 2 min). Then, place the membrane in an oven and bake it for 30 min at 80˚C. If the RNA was initially separated through a gel containing formaldehyde, this step will reverse the formaldehyde cross-linking and improve hybridization to the probe.
Prehybridization
7. Prepare 100 mL of the appropriate Church and Gilbert hybridization buffer (Church and Gilbert 1984) as outlined in Table 1. First add BSA to the H2O, and then add the remaining ingredients. After combining the ingredients, heat the buffer to 42˚C to dissolve and maintain the sodium dodecyl sulfate (SDS) in solution. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
315
T.W. Nilsen
TABLE 1. Church and Gilbert hybridization buffer Stringency High
Moderate
Low
1g 30 mL 20 mL
1g – 50 mL
1g 15 mL 50 mL
15 mL
15 mL
–
0.2 mL 35 mL
0.2 mL 35 mL
BSA H2O Sodium phosphate buffer for Church and Gilbert hybridization Formamide EDTA (0.5 M, pH 8.0) SDS (20%, w/v)
0.2 mL 35 mL
Final concentration 10 mg/mL – 0.2 M (high stringency) or 0.5 (moderate and low stringency) 15% (v/v) (high and moderate stringency) 1 mM 7% (w/v)
Use high-stringency conditions for long probes with high melting temperatures (Tm). Use moderatestringency conditions for probes that are 100 nucleotides long and made with random primers or by asymmetric polymerase chain reaction (PCR). Use low-stringency conditions for oligonucleotide probes with low Tm.
8. If pieces of the membrane are to be probed separately, cut the membrane into strips. Make orientation marks (such as diagnostic notching of corners) to distinguish the different pieces. If strips from two different gels are to be probed together, place them back to back so that the RNA side is facing out. 9. Cut vacuum seal bags large enough to leave one-half to three-quarters of an inch on either side of the membrane. 10. Seal one side of the bag. 11. Place the membrane (or membrane strips) in the bag. Position the membrane toward the bottom of the bag so that it will be easier to slide the membrane around once it is sealed in the bag. 12. Seal the second side of the bag. 13. Cut a three-quarter-inch slit in the top of the bag. 14. Add warm Church and Gilbert hybridization buffer to the bag, creating as few bubbles as possible. For a 15 × 15-cm membrane, use 20 mL of buffer. 15. Remove as many bubbles as possible and seal the top of the bag. 16. Place the bag on a rocking platform in a 42˚C incubator. Prehybridize with rocking for 3 h at 42˚C to block the nonspecific probe-binding sites on the membrane. Place the remaining hybridization buffer in the same incubator to keep the SDS in solution.
Hybridization
17. Drain the buffer from the bag. 18. Add fresh Church and Gilbert hybridization buffer (half the volume that was used for prehybridization). Add the probe to the bag gently and smoothly to avoid bubbles and spilling. 19. Seal the bag. Quarantine as many bubbles as possible by pushing the bubbles to one side of the bag and sealing them off. 20. Place the bag into a larger secondary bag and seal. If there is more than one blot, placed all the bags into the same secondary bag. This step helps to contain the radioactivity.
21. Place the secondary bag on a rotating platform in a 42˚C incubator and incubate overnight or up to 48 h. 316
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
Alternative Method for Processing Northern Blots
TABLE 2. Wash buffers and incubation temperatures
High stringency (0.2 × SSC, 0.1% SDS) Moderate stringency (0.2 × SSC, 0.1% SDS) Low stringency (2 × SSC, 1% SDS)
SSC (20×)
SDS (20%, w/v)
H2O
Temperature
20 mL
10 mL
1970 mL
65˚C
20 mL
10 mL
1970 mL
50˚C
200 mL
100 mL
1700 mL
50˚C
Washing Membrane and Visualizing Bands
22. Prepare 2 L of the wash buffer corresponding to the desired hybridization stringency as outlined in Table 2. Keep 0.6 L aside for room-temperature washes and prewarm 1.4 L to the desired hybridization temperature. 23. Pour 300 mL of wash buffer at room temperature into a plastic wash container (with lid). 24. Remove the primary bag from the secondary bag and place on a paper towel. The paper towel helps to prevent contamination of the bench paper with any radioactivity that may be on the outside of the bag.
25. Remove the blot from the primary bag and place it in the rinse container containing 300 mL of wash buffer. (Place the bag with radioactive hybridization buffer in a secondary bag and seal the secondary bag. Dispose of this bag in the radioactive waste.) Rinse and remove excess hybridization buffer from the membrane in the wash buffer with a gloved hand. The hybridization buffers are thick because of the high concentration of SDS and the residual hybridization buffer must be removed to prevent high background.
26. Pour off the wash buffer and immediately add 300 mL of fresh room-temperature wash buffer. Wash the blot on a platform shaker for 10 min at room temperature. Never let the membrane dry out. Dispose of all wash buffer in the radioactive sink.
27. Discard the room-temperature wash buffer and add 500 mL of fresh warm wash buffer to the wash container. Place the lid on the container and place the container in a shaking water bath set at the appropriate temperature for the desired stringency (see Step 22). If necessary, weigh down the container with a few lead pigs on top of a piece of Styrofoam so that it sits on and moves with the shaker’s platform. Wash for 15 min. 28. Repeat Step 27 twice (for a total of three times). 29. Remove excess liquid from the blot by placing it between two paper towels or Kimwipes. 30. Place the blot in the original orientation and wrap in plastic wrap. (If the membrane was cut into several pieces for probing, reassemble it in the original uncut orientation and make sure the marks denoting the locations of the wells are properly lined up.) 31. Expose the blot to film at –80˚C (or to a phosphorimager screen). Develop when the desired exposure has been achieved. The typical exposure time is 1–2 h for abundant messages and 16–48 h for nonabundant messages. For abundant messages, monitoring the edges of the blot with a Geiger counter should reveal little or no signal. The area at which bands are expected should be easily detectable.
RELATED INFORMATION
For the traditional method, see Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions (Rio 2015). Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
317
T.W. Nilsen
RECIPES Sodium Phosphate Buffer for Church and Gilbert Hybridization
Reagent
Quantity (for 1 L)
Na2HPO4 · 7H2O H3PO4 (85%) H2O
134 g 4 mL to 1 L
The pH of this solution must be adjusted with phosphoric acid (Church and Gilbert 1984). Store indefinitely at room temperature. SSC Buffer for Northerns (20×)
Reagent NaCl Trisodium citrate H2O
Quantity (for 1 L) 175.3 g 88.3 g to 1 L
Quantity (for 2 L) 350.6 g 176.5 g to 2 L
Final concentration (20×) 3M 0.3 M
Adjust the pH to 7.0 with NaOH or HCl. Autoclave the solution and store it at room temperature. It will last for 1 yr or longer.
REFERENCES Church GM, Gilbert W. 1984. Genomic sequencing. Proc Natl Acad Sci 81: 1991–1995. Rio DC. 2015. Northern blots: Capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot081018.
318
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
An Alternative Method for Processing Northern Blots After Capillary Transfer Timothy W. Nilsen
Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
Bovine serum albumin (BSA) EDTA (0.5 M, pH 8.0) Formamide Northern blot (i.e., a membrane containing RNA fragments that have been transferred from a gel by capillary transfer) The procedure described below is performed after capillary transfer (for capillary transfer, see Steps 1–5 in Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions [Rio 2015]).
Probe (radiolabeled, specific to the RNA of interest) SDS (20%, w/v) Sodium phosphate buffer for Church and Gilbert hybridization SSC buffer for northerns (20×) The 20× stock must be diluted for use in Step 4.
Adapted from RNA: A Laboratory Manual by Donald C. Rio, Manuel Ares Jr, Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2015 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
314
Alternative Method for Processing Northern Blots
Equipment
Aluminum foil Geiger counter (optional; see Step 31) Incubator with rotating or rocking platform (42˚C) Kimwipes (optional; see Step 29) Oven (80˚C) Paper towels Pencil Plastic container with lid to hold and mix 500 mL of liquid Plastic wrap Platform shaker at room temperature Rinse container with lid Scalpel or razor blade Scissors Styrofoam and lead pigs (optional; see Step 27) UV cross-linker with 254-nm bulbs (e.g., Stratalinker, Stratagene) Alternatively, use a 254-nm UV transilluminator (see Step 6).
Vacuum food sealer and bags Water bath (shaking) at temperature appropriate for desired stringency (see Step 22) X-ray film and developer (or phosphorimaging equipment)
METHOD Cross-Linking Perform Steps 1–5 quickly and carefully to prevent the agarose from drying and sticking to the membrane, causing unnecessary background. Do not allow the membrane to dry out at any stage during the procedure.
1. On the day after performing the capillary transfer procedure (see Steps 1–5 in Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions [Rio 2015]), remove the paper towels and the blot papers from the transfer setup. 2. Mark the locations of the wells on the membrane with a pencil. Orient the gel and the membrane by slicing off or notching a corner, using a scalpel or razor blade. 3. Peel the gel off of the membrane and discard it. 4. Rinse the membrane in 2× SSC to remove any agarose that may remain stuck to it. Gently rub the membrane with a gloved hand to be sure that the agarose is removed. 5. Place the membrane on a foil sheet with the RNA surface facing up. 6. Cross-link the RNA to the membrane by exposing the membrane to short-wave UV light at 254 nm (use the 1200-mJ auto-cross-link setting in a Stratalinker or place the membrane facedown on a 254-nm light box for 2 min). Then, place the membrane in an oven and bake it for 30 min at 80˚C. If the RNA was initially separated through a gel containing formaldehyde, this step will reverse the formaldehyde cross-linking and improve hybridization to the probe.
Prehybridization
7. Prepare 100 mL of the appropriate Church and Gilbert hybridization buffer (Church and Gilbert 1984) as outlined in Table 1. First add BSA to the H2O, and then add the remaining ingredients. After combining the ingredients, heat the buffer to 42˚C to dissolve and maintain the sodium dodecyl sulfate (SDS) in solution. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
315
T.W. Nilsen
TABLE 1. Church and Gilbert hybridization buffer Stringency High
Moderate
Low
1g 30 mL 20 mL
1g – 50 mL
1g 15 mL 50 mL
15 mL
15 mL
–
0.2 mL 35 mL
0.2 mL 35 mL
BSA H2O Sodium phosphate buffer for Church and Gilbert hybridization Formamide EDTA (0.5 M, pH 8.0) SDS (20%, w/v)
0.2 mL 35 mL
Final concentration 10 mg/mL – 0.2 M (high stringency) or 0.5 (moderate and low stringency) 15% (v/v) (high and moderate stringency) 1 mM 7% (w/v)
Use high-stringency conditions for long probes with high melting temperatures (Tm). Use moderatestringency conditions for probes that are 100 nucleotides long and made with random primers or by asymmetric polymerase chain reaction (PCR). Use low-stringency conditions for oligonucleotide probes with low Tm.
8. If pieces of the membrane are to be probed separately, cut the membrane into strips. Make orientation marks (such as diagnostic notching of corners) to distinguish the different pieces. If strips from two different gels are to be probed together, place them back to back so that the RNA side is facing out. 9. Cut vacuum seal bags large enough to leave one-half to three-quarters of an inch on either side of the membrane. 10. Seal one side of the bag. 11. Place the membrane (or membrane strips) in the bag. Position the membrane toward the bottom of the bag so that it will be easier to slide the membrane around once it is sealed in the bag. 12. Seal the second side of the bag. 13. Cut a three-quarter-inch slit in the top of the bag. 14. Add warm Church and Gilbert hybridization buffer to the bag, creating as few bubbles as possible. For a 15 × 15-cm membrane, use 20 mL of buffer. 15. Remove as many bubbles as possible and seal the top of the bag. 16. Place the bag on a rocking platform in a 42˚C incubator. Prehybridize with rocking for 3 h at 42˚C to block the nonspecific probe-binding sites on the membrane. Place the remaining hybridization buffer in the same incubator to keep the SDS in solution.
Hybridization
17. Drain the buffer from the bag. 18. Add fresh Church and Gilbert hybridization buffer (half the volume that was used for prehybridization). Add the probe to the bag gently and smoothly to avoid bubbles and spilling. 19. Seal the bag. Quarantine as many bubbles as possible by pushing the bubbles to one side of the bag and sealing them off. 20. Place the bag into a larger secondary bag and seal. If there is more than one blot, placed all the bags into the same secondary bag. This step helps to contain the radioactivity.
21. Place the secondary bag on a rotating platform in a 42˚C incubator and incubate overnight or up to 48 h. 316
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
Alternative Method for Processing Northern Blots
TABLE 2. Wash buffers and incubation temperatures
High stringency (0.2 × SSC, 0.1% SDS) Moderate stringency (0.2 × SSC, 0.1% SDS) Low stringency (2 × SSC, 1% SDS)
SSC (20×)
SDS (20%, w/v)
H2O
Temperature
20 mL
10 mL
1970 mL
65˚C
20 mL
10 mL
1970 mL
50˚C
200 mL
100 mL
1700 mL
50˚C
Washing Membrane and Visualizing Bands
22. Prepare 2 L of the wash buffer corresponding to the desired hybridization stringency as outlined in Table 2. Keep 0.6 L aside for room-temperature washes and prewarm 1.4 L to the desired hybridization temperature. 23. Pour 300 mL of wash buffer at room temperature into a plastic wash container (with lid). 24. Remove the primary bag from the secondary bag and place on a paper towel. The paper towel helps to prevent contamination of the bench paper with any radioactivity that may be on the outside of the bag.
25. Remove the blot from the primary bag and place it in the rinse container containing 300 mL of wash buffer. (Place the bag with radioactive hybridization buffer in a secondary bag and seal the secondary bag. Dispose of this bag in the radioactive waste.) Rinse and remove excess hybridization buffer from the membrane in the wash buffer with a gloved hand. The hybridization buffers are thick because of the high concentration of SDS and the residual hybridization buffer must be removed to prevent high background.
26. Pour off the wash buffer and immediately add 300 mL of fresh room-temperature wash buffer. Wash the blot on a platform shaker for 10 min at room temperature. Never let the membrane dry out. Dispose of all wash buffer in the radioactive sink.
27. Discard the room-temperature wash buffer and add 500 mL of fresh warm wash buffer to the wash container. Place the lid on the container and place the container in a shaking water bath set at the appropriate temperature for the desired stringency (see Step 22). If necessary, weigh down the container with a few lead pigs on top of a piece of Styrofoam so that it sits on and moves with the shaker’s platform. Wash for 15 min. 28. Repeat Step 27 twice (for a total of three times). 29. Remove excess liquid from the blot by placing it between two paper towels or Kimwipes. 30. Place the blot in the original orientation and wrap in plastic wrap. (If the membrane was cut into several pieces for probing, reassemble it in the original uncut orientation and make sure the marks denoting the locations of the wells are properly lined up.) 31. Expose the blot to film at –80˚C (or to a phosphorimager screen). Develop when the desired exposure has been achieved. The typical exposure time is 1–2 h for abundant messages and 16–48 h for nonabundant messages. For abundant messages, monitoring the edges of the blot with a Geiger counter should reveal little or no signal. The area at which bands are expected should be easily detectable.
RELATED INFORMATION
For the traditional method, see Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions (Rio 2015). Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
317
T.W. Nilsen
RECIPES Sodium Phosphate Buffer for Church and Gilbert Hybridization
Reagent
Quantity (for 1 L)
Na2HPO4 · 7H2O H3PO4 (85%) H2O
134 g 4 mL to 1 L
The pH of this solution must be adjusted with phosphoric acid (Church and Gilbert 1984). Store indefinitely at room temperature. SSC Buffer for Northerns (20×)
Reagent NaCl Trisodium citrate H2O
Quantity (for 1 L) 175.3 g 88.3 g to 1 L
Quantity (for 2 L) 350.6 g 176.5 g to 2 L
Final concentration (20×) 3M 0.3 M
Adjust the pH to 7.0 with NaOH or HCl. Autoclave the solution and store it at room temperature. It will last for 1 yr or longer.
REFERENCES Church GM, Gilbert W. 1984. Genomic sequencing. Proc Natl Acad Sci 81: 1991–1995. Rio DC. 2015. Northern blots: Capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions. Cold Spring Harb Protoc doi: 10.1101/pdb.prot081018.
318
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot081026
