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Northern blot analysis reveals information about RNA identity, size, and abundance, allowing a deeper understanding of gene expression levels. Our Ambion® portfolio comprises one of the industry’s most comprehensive product offerings for northern blot analysis. From Ambion® TRIzol® reagent that enables high-quality, intact RNA preparations to RNase-free reagents and 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/Yeastl RNA Isolation Kit
12183016 PureLink® RNA Micro Kit 12183555 TRIzol® Plus RNA Purification System
12183018A PureLink® RNA Mini Kit 15596026 TRIzol® Reagent

 

View our RNA Isolation Kit Selection Guide

Probe Generation

RNA probes can be produced by in vitro transcription reactions using the Ambion® 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

AM1308MMAXIscript® SP6 Kit with Manual
AM1320MMAXIscript® SP6⁄T7 Kit (15 rxns each) with Manual
AM1316MMAXIscript® T3 Kit with Manual
AM1312MMAXIscript® T7 Kit with Manual
AM1324MMAXIscript® T7⁄T3 Kit (15 rxns each) with Manual

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 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 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 Millennium™ 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 Ambion® 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 Transfering 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 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

AM8676NorthernMax® 10X Denaturing Gel Buffer
AM8552NorthernMax® Formaldehyde Load Dye (1 ml tubes)
AM1940NorthernMax® Kit
AM8672NorthernMax® One-Hour Transfer Buffer
AM8677NorthernMax® Prehybridization⁄ Hybridization Buffer
AM8678NorthernMax®-Gly 10X Gel Prep⁄Running Buffer
AM1946NorthernMax®-Gly Kit
AM8551NorthernMax®-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

AM8677NorthernMax® Prehybridization/ Hybridization Buffer
AM8670ULTRAhyb® Ultrasensitive Hybridization Buffer
AM8669ULTRAhyb® Ultrasensitive Hybridization Buffer
AM8663ULTRAhyb®-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

AM1930BrightStar® BioDetect™ Kit

Dry, Bufferless Transfer

 

 

iBlot® Dry Blotting System

  • Time savings—transfer DNA or RNA typically in 7 minutes or less
  • Reproducibility—consistent preparation and running conditions
  • Convenience—no messy buffers or laborious preparation

Learn more

TechNotes

For research use only. Not for use in diagnostic procedures.