Editing Northern blot (section)

==Procedure==
A general blotting procedure<ref name="Trayhurn1996" /> starts with extraction of total RNA from a homogenized tissue sample or from cells.  Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose [[chromatography]] to isolate only those RNAs with a [[poly(A) tail]].<ref name=Durand1993>{{cite journal | last1 = Durand | first1 = G. M. | last2 = Zukin | first2 = R. S. | year = 1993 | title = Developmental Regulation of mRNAs Encoding Rat Brain Kainate/AMPA Receptors: A Northern Analysis Study | journal = J. Neurochem. | volume = 61 | issue = 6| pages = 2239–2246 | doi = 10.1111/j.1471-4159.1993.tb07465.x | pmid = 8245974 | s2cid = 33955961 }}</ref><ref name=Mori1991>{{cite journal | last1 = Mori | first1 = H. | last2 = Takeda-Yoshikawa | first2 = Y. | last3 = Hara-Nishimura | first3 = I. | last4 = Nishimura | first4 = M. | year = 1991 | title = Pumpkin malate synthase Cloning and sequencing of the cDNA and Northern blot analysis | journal = Eur. J. Biochem. | volume = 197 | issue = 2| pages = 331–336 | doi = 10.1111/j.1432-1033.1991.tb15915.x | pmid = 1709098 }}</ref>  RNA samples are then separated by gel electrophoresis.  Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system.  [[Image:capillary blot setup.svg|thumb|Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane.]] A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them.  The transfer buffer used for the blotting usually contains [[formamide]] because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation.<ref name=Yang1993>{{cite journal | last1 = Yang | first1 = H. | last2 = McLeese | first2 = J. | last3 = Weisbart | first3 = M. | last4 = Dionne | first4 = J.-L. | last5 = Lemaire | first5 = I. | last6 = Aubin | first6 = R. A. | year = 1993 | title = Simplified high throughput protocol for Northern hybridization | journal = Nucleic Acids Research | volume = 21 | issue = 14| pages = 3337–3338 | doi = 10.1093/nar/21.14.3337 | pmid = 8341618 | pmc = 309787 }}</ref>  Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.  After a probe has been labeled, it is hybridized to the RNA on the membrane.  Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition.<ref name=Streit2009>{{cite journal | last1 = Streit | first1 = S. | last2 = Michalski | first2 = C. W. | last3 = Erkan | first3 = M. | last4 = Kleef | first4 = J. | last5 = Friess | first5 = H. | year = 2009 | title = Northern blot analysis for detection of RNA in pancreatic cancer cells and tissues | journal = Nature Protocols | volume = 4 | issue = 1| pages = 37–43 | pmid = 19131955 | doi = 10.1038/nprot.2008.216 | s2cid = 24980302 }}</ref>  The membrane is washed to ensure that the probe has bound specifically and to prevent background signals from arising.  The hybrid signals are then detected by X-ray film and can be quantified by [[densitometry]].  To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by [[microarrays]] or [[RT-PCR]].<ref name="Streit2009" />

===Gels===
[[Image:RNA agarose gel.svg|thumb|RNA run on a formaldehyde agarose gel to highlight the 28S (top band) and 18S (lower band) ribosomal subunits.]]
The RNA samples are most commonly separated on [[agarose]] gels containing [[formaldehyde]] as a denaturing agent for the RNA to limit secondary structure.<ref name="Streit2009" /><ref name=Yamanka1997>{{cite journal | last1 = Yamanaka | first1 = S. | last2 = Poksay | first2 = K. S. | last3 = Arnold | first3 = K. S. | last4 = Innerarity | first4 = T. L. | year = 1997 | title = A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme | journal = Genes Dev. | volume = 11 | issue = 3| pages = 321–333 | doi = 10.1101/gad.11.3.321 | pmid = 9030685 | doi-access = free }}</ref>  The gels can be stained with [[ethidium bromide]] (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting.<ref name="Streit2009" />  [[Polyacrylamide]] gel electrophoresis with [[urea]] can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs.<ref name=Valoczi2004>Valoczi, A., Hornyik, C., Varga, N., Burgyan, J., Kauppinen, S., Havelda, Z. (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nuc. Acids Research. 32: e175.</ref>  An RNA ladder is often run alongside the samples on an electrophoresis gel to observe the size of fragments obtained but in total RNA samples the ribosomal subunits can act as size markers.<ref name="Streit2009" />    Since the large ribosomal subunit is 28S (approximately 5kb) and the small ribosomal subunit is 18S (approximately 2kb) two prominent bands appear on the gel, the larger at close to twice the intensity of the smaller.<ref name="Streit2009" /><ref name=Gortner1996>{{cite journal | last1 = Gortner | first1 = G. | last2 = Pfenninger | first2 = M. | last3 = Kahl | first3 = G. | last4 = Weising | first4 = K. | year = 1996 | title = Northern blot analysis of simple repetitive sequence transcription in plants | journal = Electrophoresis | volume = 17 | issue = 7| pages = 1183–1189 | doi = 10.1002/elps.1150170702 | pmid = 8855401 | s2cid = 36857667 }}</ref>

===Probes===
Probes for northern blotting are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest. They can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary bases to the target sequence.<ref name="Trayhurn1996" />  RNA probes (riboprobes) that are transcribed in vitro are able to withstand more rigorous washing steps preventing some of the background noise.<ref name="Streit2009" />  Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot.<ref name=Liang1995>Liang, P. Pardee, A. B. (1995) Recent advances in differential display. Current Opinion Immunol. 7: 274–280.</ref>  The probes must be labelled either with radioactive isotopes (<sup>32</sup>P) or with [[chemiluminescence]] in which [[alkaline phosphatase]] or [[horseradish peroxidase]] (HRP) break down chemiluminescent substrates producing a detectable emission of light.<ref name=Engler-Blum1993>{{cite journal | last1 = Engler-Blum | first1 = G. | last2 = Meier | first2 = M. | last3 = Frank | first3 = J. | last4 = Muller | first4 = G. A. | year = 1993 | title = Reduction of Background Problems in Nonradioactive Northern and Southern Blot Analysis Enables Higher Sensitivity Than 32P-Based Hybridizations | journal = Anal. Biochem. | volume = 210 | issue = 2| pages = 235–244 | doi = 10.1006/abio.1993.1189 | pmid = 7685563 }}</ref>  The chemiluminescent labelling can occur in two ways: either the probe is attached to the enzyme, or the probe is labelled with a ligand (e.g. [[biotin]]) for which the ligand (e.g., [[avidin]] or [[streptavidin]]) is attached to the enzyme (e.g. HRP).<ref name="Streit2009" />  X-ray film can detect both the radioactive and chemiluminescent signals and many researchers prefer the chemiluminescent signals because they are faster, more sensitive, and reduce the health hazards that go along with radioactive labels.<ref name="Engler-Blum1993" />  The same membrane can be probed up to five times without a significant loss of the target RNA.<ref name="Yang1993" />