RNA blot (Northern)
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| - | + | The '''RNA blot''' or '''Northern blot''' (named after the [[DNA blot (Southern)]] for genomic DNA fragments) is a molecular biology technique used to separate and identify pieces of RNA. RNA molecules are separated by mass on a gel, transferred (blotted) onto a cellulose or nylon membrane, and then labelled with complementary DNA or RNA molecules. These probes are either radioactive, typically <sup>32</sup>P, or contain labelled nucleotides, e.g. DIG-dNTPs, recognisable by antibodies. RNA molecules can be detected and roughly quantified via probe hybridisation. | |
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| + | The principle of the method is nicely illustrated in this [http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture2/AMG1.14-North.gif diagram] and this [http://www.youtube.com/watch?v=KfHZFyADnNg&NR=1 video]. | ||
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| + | == Designing RNA probes == | ||
| + | * DNA probes, esp. using DIG-antibody detection, often give no/weak signal; RNA probes often better here [http://www.bio.net/bionet/mm/methods/2000-October/085274.html] | ||
| + | * minimum probe length around 25 nt (anybody has a reference for this?) [http://www.protocol-online.org/biology-forums/posts/16736.html] | ||
| + | * DNA probes may be usable for both [[qRT-PCR]] and RNA blots [http://www.protocol-online.org/biology-forums/posts/17381.html] | ||
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| + | == Lab-specific protocols == | ||
| + | * [[Endy:Northern Blot, 32P End-Labeled Probes]] | ||
| + | * [[Endy:Northern blot, AlkPhos end-labeled probes]] | ||
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| + | == See also == | ||
| + | * [[RNA]] | ||
| + | * [[RNA Extraction]] | ||
| + | * [[RNA electrophoresis]] | ||
| + | * [[BE.109:Systems engineering/Measuring DNA, RNA, protein]] | ||
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| + | == External links == | ||
| + | * [[Image:3stars.png]] [http://cancer.ucsd.edu/howelllab/Northern.html RNA blot protocol for cells in culture, complete with materials] (1999) by Howell lab, UCSD | ||
| + | * [[Image:3stars.png]] [http://www.bio.net/bionet/mm/methods/2000-October/085274.html detailed RNA blot protocol for DIG probes] (2000) by Arnoud van Vliet | ||
| + | * [[Image:2stars.png]] [http://www.protocol-online.org/cgi-bin/htsearch?config=htdig_pol&restrict=&exclude=&words=northern+blot archived protocol conversations from Protocol Online] | ||
| + | * [[Image:2stars.png]] [http://www.protocol-online.org/prot/Molecular_Biology/RNA/Northern_Blotting/ index of RNA blot protocols from Protocol Online] | ||
| + | * [[Image:2stars.png]] [http://kellylab.wustl.edu/protocols/RNA_and_northern_blot3-3-2006.pdf RNA preparation and blotting protocol] (2006) by Kelly lab, Washington Uni | ||
| + | * [[Image:2stars.png]] [http://biology.semo.edu/agathman/research/protocols/northern.htm RNA blot protocol] by Allen Gathman from Southeast Missouri State Uni | ||
| + | * [[Image:2stars.png]] [http://www.ambion.com/techlib/basics/northerns/ The basics of RNA blots], Ambion general article | ||
| + | * [[Image:1stars.png]] [http://www.ambion.com/techlib/tn/63/631.html Techniques to detect mRNA - includes RNA blot], Ambion TechNotes | ||
| + | * [[Image:1stars.png]] [http://www.roche-applied-science.com/pack-insert/2039672a.pdf Pack insert DIG RNA detection kit], Roche | ||
| + | * [http://www.mcb.arizona.edu/parker/PROTOCOLS/NorthernOligoHyb.htm Probe hybridization protocol] - Parker lab (don't know how many stars to give it!) | ||
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| + | [[Category:Protocol]] | ||
| + | [[Category:RNA]] | ||
| + | [[Category:In vitro]] | ||
Current revision
The RNA blot or Northern blot (named after the DNA blot (Southern) for genomic DNA fragments) is a molecular biology technique used to separate and identify pieces of RNA. RNA molecules are separated by mass on a gel, transferred (blotted) onto a cellulose or nylon membrane, and then labelled with complementary DNA or RNA molecules. These probes are either radioactive, typically 32P, or contain labelled nucleotides, e.g. DIG-dNTPs, recognisable by antibodies. RNA molecules can be detected and roughly quantified via probe hybridisation.
The principle of the method is nicely illustrated in this diagram and this video.
Contents |
Designing RNA probes
- DNA probes, esp. using DIG-antibody detection, often give no/weak signal; RNA probes often better here [1]
- minimum probe length around 25 nt (anybody has a reference for this?) [2]
- DNA probes may be usable for both qRT-PCR and RNA blots [3]
Lab-specific protocols
See also
External links
-
RNA blot protocol for cells in culture, complete with materials (1999) by Howell lab, UCSD
- detailed RNA blot protocol for DIG probes (2000) by Arnoud van Vliet
-
archived protocol conversations from Protocol Online
- index of RNA blot protocols from Protocol Online
- RNA preparation and blotting protocol (2006) by Kelly lab, Washington Uni
- RNA blot protocol by Allen Gathman from Southeast Missouri State Uni
- The basics of RNA blots, Ambion general article
-
Techniques to detect mRNA - includes RNA blot, Ambion TechNotes
- Pack insert DIG RNA detection kit, Roche
- Probe hybridization protocol - Parker lab (don't know how many stars to give it!)
