RNA blot (Northern)

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The '''RNA blot''' or '''Northern blot''' (named after the [[Southern blot]] for 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 '''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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For a schematic overview of the method see [http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture2/AMG1.14-North.gif here].
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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].
== Designing RNA probes ==
== Designing RNA probes ==
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* minimum probe length around 25 nt (anybody has a reference for this?) [http://www.protocol-online.org/biology-forums/posts/16736.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]
* 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 ==
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* [[Endy:Northern Blot, 32P End-Labeled Probes]]
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* [[Endy:Northern blot, AlkPhos end-labeled probes]]
== See also ==
== See also ==
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* [[RNA Extraction]]
* [[RNA Extraction]]
* [[RNA electrophoresis]]
* [[RNA electrophoresis]]
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* [[Endy:Northern Blot, 32P End-Labeled Probes]]
 
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* [[Endy:Northern blot, AlkPhos end-labeled probes]]
 
* [[BE.109:Systems engineering/Measuring DNA, RNA, protein]]
* [[BE.109:Systems engineering/Measuring DNA, RNA, protein]]
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* [[Image:1stars.png]] [http://www.roche-applied-science.com/pack-insert/2039672a.pdf Pack insert DIG RNA detection kit], Roche
* [[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!)
* [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:Protocol]]
[[Category:RNA]]
[[Category:RNA]]
[[Category:In vitro]]
[[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

Personal tools