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Northern Blotting
Northern blot analysis reveals information about RNA identity, size, and abundance, allowing a deeper understanding of gene expression levels. Our Invitrogen™ portfolio comprises one of the industry’s most comprehensive product offerings for northern blot analysis. From Invitrogen™ TRIzol™ reagent that enables high-quality, intact RNA preparations to RNase-free reagents and Invitrogen™ BrightStar™-Plus membranes, this array of products meets all your Northern blot analysis needs and helps shorten the time it takes you to get results.
Step-by-Step Guide to Northern Blot Analysis
RNA Isolation
Obtaining high-quality, intact RNA is a critical step in performing northern blot analysis. All protocols, techniques, and commercially available kits used to isolate RNA share these common attributes:
- Cellular lysis and membrane disruption
- Inhibition of ribonuclease activity
- Deproteinization
- Recovery of intact RNA
Products for RNA Isolation
| IS10008 | iPrep™ PureLink® Virus Kit | K156001 | PureLink® Total RNA Blood Kit |
| AM1934 | LeukoLOCK™ Total RNA Isolation System | 12280-050 | PureLink® Viral RNA/DNA Mini Kit |
| AM1840 | MagMAX™ Total Nucleic Acid Isolation Kit | AM1975 | RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE |
| AM1839 | MagMAX™-96 for Microarrays Total RNA Isolation Kit | AM1925 | RiboPure™ -Bacteria/Yeast Kit |
| AM1830 | MagMAX™-96 Total RNA Isolation Kit | AM1926 | RiboPure™ -Yeast Kit |
| AM1936 | MagMAX™-96 Viral RNA Isolation Kit | AM1928 | RiboPure™-Blood Kit |
| AM1983 | MELT™ Total Nucleic Acid Isolation System | AM1912 | RNAqueous® Kit |
| AM9690 | Plant RNA Isolation Aid | AM1931 | RNAqueous® -Micro Kit |
| 12322012 | Plant RNA Reagent | 4380204 | Tempus® Spin RNA Isolation Kit |
| 12173011A | PureLink® Pro 96 Total RNA Purification Kit | 10296010 | TRIzol® LS Reagent |
| 122800-96A | PureLink® Pro 96 Viral RNA/DNA Purification Kit | 16096-020 | TRIzol® Max™ Bacteria/Yeast RNA Isolation Kit |
| 12183016 | PureLink® RNA Micro Kit | 12183555 | TRIzol® Plus RNA Purification System |
| 12183018A | PureLink® RNA Mini Kit | 15596026 | TRIzol® Reagent |
Probe Generation
RNA probes can be produced by in vitro transcription reactions using the Invitrogen™ MAXIscript™ Kit. Isotope- or nonisotope-labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations.
Technical Considerations:
When performing northern blot analyses using RNA probes rather than DNA probes, we recommend that several adjustments be made to obtain the best results. These include:
- The type of membrane used
- RNA-appropriate hybridization buffer
- Elevated hybridization and wash temperatures
- Probe techniques.
Products for Probe Generation
Denaturing
Once RNA samples are isolated, the next step in northern blot analysis is denaturing agarose gel electrophoresis. Formaldehyde has been the denaturant traditionally used during electrophoresis. The Invitrogen™ NorthernMax™ kit contains a complete set of RNase-free reagents for running formaldehyde-based agarose gels. The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood. With the Invitrogen™ NorthernMax™-Gly Kit, RNA samples are denatured in glyoxal/DMSO and run without the safety issues associated with formaldehyde.
A wide range of RNA ladders are available for accurate mRNA size and mass estimations, including the Invitrogen™ Millennium™ RNA Markers. Go to RNA ladders for more information.
Products for Denaturing
| AM7785 | Millennium™ Marker Probe Template | 750018 | 50x Denhardt’s solution |
| AM8676 | NorthernMax® 10X Denaturing Gel Buffer | 750023 | UltraPure™ DEPC-treated water |
| AM8552 | NorthernMax® Formaldehyde Load Dye (1 mL tubes) | 10977-015 | UltraPure™ DNAse/RNase Free Distilled water |
| AM1940 | NorthernMax® Kit | 16550100 | UltraPure™ Agarose 1000 |
| AM8672 | NorthernMax® One-Hour Transfer Buffer | 14476028 | UltraPure™ 0.5M EDTA, pH 8.0 |
| AM8677 | NorthernMax® Prehybridization⁄ Hybridization Buffer | 24740011 | UltraPure™ 5M NaCl |
| AM8678 | NorthernMax®-Gly 10X Gel Prep⁄Running Buffer | 15506017 | UltraPure™ Tris HCl |
| AM1946 | NorthernMax®-Gly Kit | 15557036 | UltraPure™ 20X SSC |
| AM8551 | NorthernMax®-Gly Sample Loading Dye (1 mL tubes) | 15591035 | UltraPure™ 20X SSPE |
Transfer to Solid Support and Immobilization
Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization. For fast, reproducible transfer, the iBlot Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.
Alternatively, we offer a rapid alkaline transfer method that is incorporated into the NorthernMax and NorthernMax-Gly procedure. Instead of overnight transfer, the transfer takes about 2 hours and delivers higher blot sensitivity by efficiently moving RNA (especially larger transcripts) onto the membrane. The NorthernMax and NorthernMax-Gly Kits have been optimized to work with the BrightStar-Plus positively charged nylon membranes, and we recommend their use to minimize background and maximize signal. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer and should not be used with these kits.
Products for Transferring to Solid Support and Immobilization
| AM10100 | BrightStar®-Plus Positively Charged Nylon Membrane (15 x 15 cm) | ||
| AM10104 | BrightStar®-Plus Positively Charged Nylon Membrane (large roll, 30 cm x 3 m) | ||
| AM10102 | BrightStar®-Plus Positively Charged Nylon Membrane (small roll, 30 x 45 cm) | ||
| 15515-025 | UltraPure™ Formamide | ||
| AM9342 | Formamide (Deionized) | ||
| LC2003 | Novex® Nylon Positively Charged Membrane, 045 micron (8.3 x 7.3 cm) | ||
Prehybridization and Hybridization with Probe
The Invitrogen™ NorthernMax™ and NorthernMax™-Gly kits include ULTRAhyb™ Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb™ buffer and then added to the prehybridized blot. ULTRAhyb buffer can increase sensitivity up to 100-fold compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb buffer maximizes blot sensitivity, typically hybridization can be performed in just 2 hours for many messages.
Products for Prehybridization and Hybridization with Probe
| AM8676 | NorthernMax® 10X Denaturing Gel Buffer | ||
| AM8552 | NorthernMax® Formaldehyde Load Dye (1 ml tubes) | ||
| AM1940 | NorthernMax® Kit | ||
| AM8672 | NorthernMax® One-Hour Transfer Buffer | ||
| AM8677 | NorthernMax® Prehybridization⁄ Hybridization Buffer | ||
| AM8678 | NorthernMax®-Gly 10X Gel Prep⁄Running Buffer | ||
| AM1946 | NorthernMax®-Gly Kit | ||
| AM8551 | NorthernMax®-Gly Sample Loading Dye (1 ml tubes) | ||
Washing
After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized molecules. Certified RNase-free low- and high-stringency wash buffers are included in the NorthernMax Kits, and are also available separately.
Products for Washing
| AM8677 | NorthernMax® Prehybridization/ Hybridization Buffer | ||
| AM8670 | ULTRAhyb® Ultrasensitive Hybridization Buffer | ||
| AM8669 | ULTRAhyb® Ultrasensitive Hybridization Buffer | ||
| AM8663 | ULTRAhyb®-Oligo | ||
Detection
The BrightStar™ BioDetect™ Nonisotopic Detection Kit provides all the reagents and materials necessary for detection of biotinylated RNA and DNA probes. The NorthernMax Kits have been optimized in conjunction with the BrightStar BioDetect Kit for ultrasensitive nonisotopic northern blots with a high signal-to-noise ratio and low background. This optimization of the northern blotting method also yields excellent results with radiolabeled probes.
Products for Detection
| AM1930 | BrightStar® BioDetect™ Kit |
RNA Isolation
Obtaining high-quality, intact RNA is a critical step in performing northern blot analysis. All protocols, techniques, and commercially available kits used to isolate RNA share these common attributes:
- Cellular lysis and membrane disruption
- Inhibition of ribonuclease activity
- Deproteinization
- Recovery of intact RNA
Products for RNA Isolation
| IS10008 | iPrep™ PureLink® Virus Kit | K156001 | PureLink® Total RNA Blood Kit |
| AM1934 | LeukoLOCK™ Total RNA Isolation System | 12280-050 | PureLink® Viral RNA/DNA Mini Kit |
| AM1840 | MagMAX™ Total Nucleic Acid Isolation Kit | AM1975 | RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE |
| AM1839 | MagMAX™-96 for Microarrays Total RNA Isolation Kit | AM1925 | RiboPure™ -Bacteria/Yeast Kit |
| AM1830 | MagMAX™-96 Total RNA Isolation Kit | AM1926 | RiboPure™ -Yeast Kit |
| AM1936 | MagMAX™-96 Viral RNA Isolation Kit | AM1928 | RiboPure™-Blood Kit |
| AM1983 | MELT™ Total Nucleic Acid Isolation System | AM1912 | RNAqueous® Kit |
| AM9690 | Plant RNA Isolation Aid | AM1931 | RNAqueous® -Micro Kit |
| 12322012 | Plant RNA Reagent | 4380204 | Tempus® Spin RNA Isolation Kit |
| 12173011A | PureLink® Pro 96 Total RNA Purification Kit | 10296010 | TRIzol® LS Reagent |
| 122800-96A | PureLink® Pro 96 Viral RNA/DNA Purification Kit | 16096-020 | TRIzol® Max™ Bacteria/Yeast RNA Isolation Kit |
| 12183016 | PureLink® RNA Micro Kit | 12183555 | TRIzol® Plus RNA Purification System |
| 12183018A | PureLink® RNA Mini Kit | 15596026 | TRIzol® Reagent |
Probe Generation
RNA probes can be produced by in vitro transcription reactions using the Invitrogen™ MAXIscript™ Kit. Isotope- or nonisotope-labeled nucleotides can be incorporated directly during synthesis with this kit, or the RNA can be synthesized unlabeled and subsequently treated with Psoralen-Biotin to produce biotinylated probes for blot hybridizations.
Technical Considerations:
When performing northern blot analyses using RNA probes rather than DNA probes, we recommend that several adjustments be made to obtain the best results. These include:
- The type of membrane used
- RNA-appropriate hybridization buffer
- Elevated hybridization and wash temperatures
- Probe techniques.
Products for Probe Generation
Denaturing
Once RNA samples are isolated, the next step in northern blot analysis is denaturing agarose gel electrophoresis. Formaldehyde has been the denaturant traditionally used during electrophoresis. The Invitrogen™ NorthernMax™ kit contains a complete set of RNase-free reagents for running formaldehyde-based agarose gels. The disadvantage of using formaldehyde is the need to pour and run gels in a fume hood. With the Invitrogen™ NorthernMax™-Gly Kit, RNA samples are denatured in glyoxal/DMSO and run without the safety issues associated with formaldehyde.
A wide range of RNA ladders are available for accurate mRNA size and mass estimations, including the Invitrogen™ Millennium™ RNA Markers. Go to RNA ladders for more information.
Products for Denaturing
| AM7785 | Millennium™ Marker Probe Template | 750018 | 50x Denhardt’s solution |
| AM8676 | NorthernMax® 10X Denaturing Gel Buffer | 750023 | UltraPure™ DEPC-treated water |
| AM8552 | NorthernMax® Formaldehyde Load Dye (1 mL tubes) | 10977-015 | UltraPure™ DNAse/RNase Free Distilled water |
| AM1940 | NorthernMax® Kit | 16550100 | UltraPure™ Agarose 1000 |
| AM8672 | NorthernMax® One-Hour Transfer Buffer | 14476028 | UltraPure™ 0.5M EDTA, pH 8.0 |
| AM8677 | NorthernMax® Prehybridization⁄ Hybridization Buffer | 24740011 | UltraPure™ 5M NaCl |
| AM8678 | NorthernMax®-Gly 10X Gel Prep⁄Running Buffer | 15506017 | UltraPure™ Tris HCl |
| AM1946 | NorthernMax®-Gly Kit | 15557036 | UltraPure™ 20X SSC |
| AM8551 | NorthernMax®-Gly Sample Loading Dye (1 mL tubes) | 15591035 | UltraPure™ 20X SSPE |
Transfer to Solid Support and Immobilization
Once separated by denaturing agarose gel electrophoresis, the RNA is transferred to a positively charged nylon membrane and immobilized for subsequent hybridization. For fast, reproducible transfer, the iBlot Dry Blotting System offers complete transfer of RNA to nylon membrane typically in 7 minutes. With dry blotting, there is no need for additional buffer or liquids that can introduce variability into the result.
Alternatively, we offer a rapid alkaline transfer method that is incorporated into the NorthernMax and NorthernMax-Gly procedure. Instead of overnight transfer, the transfer takes about 2 hours and delivers higher blot sensitivity by efficiently moving RNA (especially larger transcripts) onto the membrane. The NorthernMax and NorthernMax-Gly Kits have been optimized to work with the BrightStar-Plus positively charged nylon membranes, and we recommend their use to minimize background and maximize signal. Please note that nitrocellulose membranes are chemically incompatible with the NorthernMax Transfer Buffer and should not be used with these kits.
Products for Transferring to Solid Support and Immobilization
| AM10100 | BrightStar®-Plus Positively Charged Nylon Membrane (15 x 15 cm) | ||
| AM10104 | BrightStar®-Plus Positively Charged Nylon Membrane (large roll, 30 cm x 3 m) | ||
| AM10102 | BrightStar®-Plus Positively Charged Nylon Membrane (small roll, 30 x 45 cm) | ||
| 15515-025 | UltraPure™ Formamide | ||
| AM9342 | Formamide (Deionized) | ||
| LC2003 | Novex® Nylon Positively Charged Membrane, 045 micron (8.3 x 7.3 cm) | ||
Prehybridization and Hybridization with Probe
The Invitrogen™ NorthernMax™ and NorthernMax™-Gly kits include ULTRAhyb™ Ultrasensitive Hybridization Buffer, which can be used for both prehybridization and hybridization. Although double-stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA probes can be diluted in a small amount of ULTRAhyb™ buffer and then added to the prehybridized blot. ULTRAhyb buffer can increase sensitivity up to 100-fold compared to other hybridization solutions by pushing hybridization to completion without increasing background. As few as 10,000 molecules can be detected. Because ULTRAhyb buffer maximizes blot sensitivity, typically hybridization can be performed in just 2 hours for many messages.
Products for Prehybridization and Hybridization with Probe
| AM8676 | NorthernMax® 10X Denaturing Gel Buffer | ||
| AM8552 | NorthernMax® Formaldehyde Load Dye (1 ml tubes) | ||
| AM1940 | NorthernMax® Kit | ||
| AM8672 | NorthernMax® One-Hour Transfer Buffer | ||
| AM8677 | NorthernMax® Prehybridization⁄ Hybridization Buffer | ||
| AM8678 | NorthernMax®-Gly 10X Gel Prep⁄Running Buffer | ||
| AM1946 | NorthernMax®-Gly Kit | ||
| AM8551 | NorthernMax®-Gly Sample Loading Dye (1 ml tubes) | ||
Washing
After hybridization, unhybridized probe is removed by washing in several changes of buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove partially hybridized molecules. Certified RNase-free low- and high-stringency wash buffers are included in the NorthernMax Kits, and are also available separately.
Products for Washing
| AM8677 | NorthernMax® Prehybridization/ Hybridization Buffer | ||
| AM8670 | ULTRAhyb® Ultrasensitive Hybridization Buffer | ||
| AM8669 | ULTRAhyb® Ultrasensitive Hybridization Buffer | ||
| AM8663 | ULTRAhyb®-Oligo | ||
Detection
The BrightStar™ BioDetect™ Nonisotopic Detection Kit provides all the reagents and materials necessary for detection of biotinylated RNA and DNA probes. The NorthernMax Kits have been optimized in conjunction with the BrightStar BioDetect Kit for ultrasensitive nonisotopic northern blots with a high signal-to-noise ratio and low background. This optimization of the northern blotting method also yields excellent results with radiolabeled probes.
Products for Detection
| AM1930 | BrightStar® BioDetect™ Kit |
Dry, Bufferless Transfer
| iBlot Dry Blotting System
|
TechNotes
- TechNotes 6(3): Strategies for Detecting mRNA: A comparison of northern blotting, nuclease protection assays, in situ hybridization, and RT-PCR for detecting mRNA.
- TechNotes 7(3): Tools for Blot Hybridization: Maximize sensitivity, increase data quality, and increase blot lifespans.
- TechNotes 8(4): Stable Markers for Northern Analysis: Markers provided in formamide for increased RNase resistance.
- TechNotes 9(1): Premade, RNase-free Northern Reagents: Increase northern sensitivity by limiting degradation of probe and of RNA samples bound to your blots.
- TechNotes 9(2): Detect Rare Transcripts: Reduce northern blot exposure times from days to hours by pushing blot hybridizations toward completion.
- TechNotes 11(2): Northern Analysis: Faster and Better: Decrease hybridization and exposure time with ULTRAhyb Ultrasensitive Hybridization Buffer.
- TechNotes 14(2): Get More From Your Blots with ULTRAhyb Technology: Here we show that by selecting a more sensitive hybridization buffer, the percentage of target molecules that hybridize to probe on a northern blot can be significantly increased.
Technical Articles
For Research Use Only. Not for use in diagnostic procedures.
Stylesheet for Classic Wide Template adjustments