Sponsor / Advertise | Link to us | Contact us | About us | Help us

Molecular Biology Blog

Recent Forum Posts

 

Northern Blot

Copyright 2007 - Molecular Station

AUDIO Listen to a detailed Northern Blot Explanation!

History of Northern Blotting

Northern blotting is an RNA blotting technique which was developed in 1977 by Alwine et al. at Stanford University (ref:1). It was named after the Southern blot technique which blots for DNA, invented by Edwin M. Southern in 1975. Western blot, a method for blotting for proteins is also a play on the Southern blot naming and was named in 1981 (ref:2).

Background Information - Northern Blot Technique

Northern analysis despite its age in the high tech world of Real Time PCR, nuclease protection assays (RPAs) and microarrays, is still the gold-standard for the detection and quantitation of mRNA levels. This is because northern blot analysis allows a direct comparison of the messenger RNA abundance between samples on a single membrane.

In northern blot the main difference between the other blotting techniques is that RNA is the factor being detected. Also, due to the fact that RNA is usually single-stranded, it creates complex secondary structures which affect its migration and hence denaturing conditions are used to run the gels (unlike Southern).

RNA is separated out by RNA gel electrophoresis (usually agarose gel electrophoresis), subsequent transfer to membrane, hybridization with probe, and finally detection.

 

 

Similarly to Southern blotting, the hybridization probes may be DNA or RNA in northern blotting.

A variant of the procedure known as the reverse northern blot was occasionally (although, infrequently) used. In this procedure, the substrate nucleic acid (that is affixed to the membrane) is a collection of isolated DNA fragments, and the probe is RNA extracted from a tissue and radioactively labelled.

The use of DNA microarrays that have come into widespread use in the late 1990s and early 2000s is more akin to the reverse procedure, in that they involve the use of isolated DNA fragments affixed to a substrate, and hybridization with a probe made from cellular RNA. Thus the reverse procedure, though originally uncommon, enabled the one-at-a-time study of gene expression using northern analysis to evolve into gene expression profiling, in which many (possibly all) of the genes in an organism may have their expression monitored

Applications of the Northern Blot

Northern blots have been superceded in most areas by Real Time PCR and microarray approaches. It is not often used for clinical or diagnostic purposes.

The northern blot protocol and its variations are used however in molecular biology research to:

• a gold-standard for the direct study of gene expression at the level of mRNA (messenger RNA transcripts).
• detection of mRNA transcript size
• study RNA degradation
• study RNA splicing - can detect alternatively spliced transcripts
• study RNA half-life
• study IRES (internal ribosomal entry site) - to remove possibility of RNA digestion vs 2nd cistron translation.
• often used to confirm and check transgenic / knockout mice (animals)

Disadvantages of Northern Blotting

The disadvantages of using northern blotting include:

• Often radioactivity is used. This prevents ease of performing it, use and disposal. New methods of non-radioactive detection have been generated allowing non-radioactive detection. See Pierce.
• The whole process of northern blotting takes a long time usually, from sample preparation through to detection.
• If RNA samples are even slightly degraded by RNases, the quality of the data and quantitation of expression is quite negatively affected.
• The standard northern blot method is relatively less sensitive than nuclease protection assays and RT-PCR. The sensitivity of northern blots may be increased with the use of nylon positively-charged membranes, use of a highly specific antisense probe.
• Detection with multiple probes is a problem. Often, the membranes must be stripped before hybridization and detection with a second probe. This is a problem as harsh conditions are required to strip off probes from the blot and is also time consuming. Also, there is a limit to the amount of times a blot may be stripped.

Advantages of Northern Blotting

The advantages of northern blots include:

• It is a widely accepted and well regarded method
• northern blotting is a straight-forward method
• Often it is used as a confirmation or check
• Often a gold-standard
• it is a versatile protocol as it can allow the usage of many types of probes (vs Real time PCR) including: radiolabeled and non-radiolabeled, in vitro transcribed RNA and even oligonucleotides such as primers.
• Sequences with even partial homology, unlike real time PCR or other methods can be used as hybridization probes (i.e sequence from different species for homology analysis, or even genomic fragments can be used).

Northern Blot Protocol

As mentioned the steps in northern blotting include:

1. RNA isolation
2. Gel electrophoresis of RNA for separation
3. Transfer to membrane (usually positively charged nylon as RNA is negatively charged)
4. Cross-linking of RNA to membrane (usually by UV-crosslinking or chemical means)
5. Hybridization
6. Detection

Materials Needed for Northern Blot Protocol

Buffer Recipes

20XSSPE (500 ml)

• 3.6M NaCl : 105.2 g
• 0. 2M phosphate buffer pH 7.0: 100mL of 1M
• 20mM EDTA: 20mL of 0.5M

Phosphate buffer pH7.0 (500ml)

• NaH2PO4 (monobasic) 140 mL of 1M
• Na2H2PO4 (Dibasic) 360 mL of 1M
• pH to 7.0 after both are added

Hybridization buffer =HB (100mls)

• 5X SSPE: 25mls 20X
• 2% SDS: 20 mls 10%

 

* *Right before using add 1000ug denatured RNAse free CT DNA (heat to 95o for 3 min to denature) 5000ug yeast RNA

 

WASH: 2X SSPE + 0.1% SDS (500 mls) 50 mls 20X 5 mls 10% SDS= 0.1% SDS

Running buffer for RNA gel (1L)

• 100 mls 10X MOPS
• 52. 6 mls of formaldehyde
• 847. 4 mls H20

RNA Gel (70ml)

• 0.84 g agarose
• 7 mls 10x MOPS
• 58.38 mls H2O
• Put gel in solution by heating. Let gel cool to 60°C, then add Formaldehyde to equal 0.66M --3.78 mls
• Pour gel in fume hood if possible

RNA Samples for Northern Blot

See RNA Isolation and Purification

RNA Gel

After isolating RNA, the RNA must be separated out on a gel (usually agarose).

see RNA gels

Northern Blot Transfer to Nylon Membrane Protocol

After running the RNA gel, wash the gel with distilled water put on posiblotter.

Layers from top to bottom are:

• sponge
• 3-4 Whatman papers cut to size ( both of these up above should be soaked in 10X SSPE but not dripping)
• gel mask.
• Note: Make sure the gel overlays the mask so that it can seal membrane with line of the top of gel and the right top corner marked 3 pieces slightly bigger 3M paper hard plastic with holes

Clamp on and make sure there is a good seal. Use 75-80 mm Hg of pressure for 1 hr or more.

Blot membrane dry

Cross-linking Nylon Membranes - Northern Blot Protocol

• Cut top right corner wrap in saran wrap.
• UV irradiate with RNA side down for 2.5 min.
• Wash for a couple of minutes in hot 2X SSPE/0.1% SDS solution (Microwave solution for 30-45 seconds at 50% power.
• Air dry

Pre Hybridization for Northern Blot

1. Pre Hybridize for 6 hours-overnight
2. Set oven to 65°C heat HB buffer to put SDS in solution
3. Roll up membrane inside piece of mesh and put into hybridizaton tube(2X SSPE/0.1%SDS)
4. Check for air bubbles
5. Put tube in oven balanced with another tube on opposite side

Labeling probe for Northern Blotting

1. Put 25ng of probe in a total volume of 11uL with ddH2O
2. Denature @ 95°C 3 min. This is to get the probe single stranded and remove secondary structure which would prevent efficient hybridization.
3. Quick cool on wet ice. This is to keep the probe single stranded, free of secondary structure and not allow reannealing (or reforming of secondary structures).
4. Add 4ul High Prime (Boehringer Mannheim) 5ul alpha radiolabeled P-32 dCTP 3000uCi/mmole 20ul total
5. Incubate 10-15 min 37°C
6. Add 28uL of 1X TE, 2ul 0.5M EDTA, to 20ul reaction.
7. Pre-spin G-25 Sephadex column for 1 minute.
8. Add 50ul sample to column and spin for 2.5 min in clinical centrifuge. This is to purify probe away from label.
9. Throw away column.
10. Mix in a new tube 100ug CT-DNA 50ug Yeast RNA in a 0.5mL tube.
11. Denature 95° 3 min
12. Add to hybrid tube mix, hybridize at 65°C for 20-36 hrs

Post hybridization Protocol for Northern Blotting

Hybridize for 36-48 hours then wash.

1. Heat up 2X SSPE/0.1% SDS (wash) heat up to 65° C (Use microwave or water bath to warm solution)
2. Take out tube and pour liquid into hot radioactive waste container.
3. Rinse with 10ml wash do this twice and pour waste out.
4. Put in 40 to 50 mls of wash and shake vigoursly.
5. Put in hybridization oven for 20 min rotating.
6. Pour down sink
7. Another 40-50mls and shake in oven.
8. Pour out to waste container (sink if not hot or toxic) and make sure it is no longer hot and then take out membrane.
9. Wash on shaker with 0.2XSSPE w/ 0.1% SDS at RT for 15 min.
10. Air dry
11. Expose

 

Tips for Northern Blot

If you currently have a preferred method for isolating RNA, continue to use it.

Always run an agarose gel of your RNA to assess the quality. Do not only rely on spectrophotometry results.

* Use DEPC treated water and baked glassware. This solution, absolutely, positively must be Rnase free. Rnase is everywhere! Wear gloves, use only baked glassware, or virgin plastic. DePC treat your water, buffers (except Tris). Be very careful.

 

Troubleshooting Northern Blots

If you currently have a preferred method for isolating RNA, continue to use it.

 

Discuss and post questions about this in the Northern Blot Forum.

Northern Blot References

1. Alwine JC, Kemp DJ, Stark GR (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 (12): 5350-4.
2. W. Neal Burnette (April 1981). "'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate — polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A". Anal Biochem 112 (2): 195-203.