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tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organs. If an upregulated gene is observed by an abundance of mRNA on the northern blot the sample can then be sequenced to determine if the gene is known to researchers or if it is a novel finding. The expression patterns obtained under given conditions can provide insight into the function of that gene. Since the RNA is first separated by size, if only one probe type is used variance in the level of each band on the membrane can provide insight into the size of the product, suggesting alternative splice products of the same gene or repetitive sequence motifs. The variance in size of a gene product can also indicate deletions or errors in transcript processing. By altering the probe target used along the known sequence it is possible to determine which region of the RNA is missing.
230:, serial analysis of gene expression (SAGE), as well as northern blotting. Microarrays are quite commonly used and are usually consistent with data obtained from northern blots; however, at times northern blotting is able to detect small changes in gene expression that microarrays cannot. The advantage that microarrays have over northern blots is that thousands of genes can be visualized at a time, while northern blotting is usually looking at one or a small number of genes.
127:
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. 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
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209:) is attached to the enzyme (e.g. HRP). 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. The same membrane can be probed up to five times without a significant loss of the target RNA.
238:). The chemicals used in most northern blots can be a risk to the researcher, since formaldehyde, radioactive material, ethidium bromide, DEPC, and UV light are all harmful under certain exposures. Compared to RT-PCR, northern blotting has a low sensitivity, but it also has a high specificity, which is important to reduce false positive results.
184:
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. RNA probes (riboprobes) that are transcribed in vitro are able to
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can also be used in RNA separation but it is most commonly used for fragmented RNA or microRNAs. 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. Since the
73:
complementary to part of or the entire target sequence. Strictly speaking, the term 'northern blot' refers specifically to the capillary transfer of RNA from the electrophoresis gel to the blotting membrane. However, the entire process is commonly referred to as northern blotting. The northern blot
260:
Researchers occasionally use a variant of the procedure known as the reverse northern blot. 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
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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,
126:
because it lowers the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation. 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
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The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years
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Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment. The technique has been used to show overexpression of oncogenes and downregulation of
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withstand more rigorous washing steps preventing some of the background noise. Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot. The probes must be labelled either with radioactive isotopes (P) or with
110:. 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.
233:
A problem in northern blotting is often sample degradation by RNases (both endogenous to the sample and through environmental contamination), which can be avoided by proper sterilization of glassware and the use of RNase inhibitors such as DEPC
197:(HRP) break down chemiluminescent substrates producing a detectable emission of light. 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.
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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
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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:
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Engler-Blum, G.; Meier, M.; Frank, J.; Muller, G. A. (1993). "Reduction of
Background Problems in Nonradioactive Northern and Southern Blot Analysis Enables Higher Sensitivity Than 32P-Based Hybridizations".
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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.
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Baldwin, D., Crane, V., Rice, D. (1999) A comparison of gel-based, nylon filter and microarray techniques to detect differential RNA expression in plants. Current
Opinion in Plant Biol. 2: 96β103.
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A general blotting procedure 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
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the nonradioactive technique was compared to a radioactive technique and found as sensitive as the radioactive one, but requires no protection against radiation and is less time-consuming.
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Kevil, C. G., Walsh, L., Laroux, F. S., Kalogeris, T., Grisham, M. B., Alexander, J. S. (1997) An
Improved, Rapid Northern Protocol. Biochem. and Biophys. Research Comm. 238:277β279.
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Kreft, K., Kreft, S., Komel, R., GrubiΔ, Z. (2000). Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. PflΓΌgers Arch β Eur J Physiol, 439:R66-R67
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Alberts, B., Johnson, A., Lewis, J. Raff, M., Roberts, K., Walter, P. 2008. Molecular
Biology of the Cell, 5th ed. Garland Science, Taylor & Francis Group, NY, pp 538β539.
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With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression rates during differentiation and
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Mori, H.; Takeda-Yoshikawa, Y.; Hara-Nishimura, I.; Nishimura, M. (1991). "Pumpkin malate synthase
Cloning and sequencing of the cDNA and Northern blot analysis".
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Streit, S.; Michalski, C. W.; Erkan, M.; Kleef, J.; Friess, H. (2009). "Northern blot analysis for detection of RNA in pancreatic cancer cells and tissues".
704:"A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA-editing enzyme"
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Taniguchi, M.; Miura, K.; Iwao, H.; Yamanaka, S. (2001). "Quantitative
Assessment of DNA Microarrays β Comparison with Northern Blot Analysis".
131:. To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by
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Durand, G. M.; Zukin, R. S. (1993). "Developmental
Regulation of mRNAs Encoding Rat Brain Kainate/AMPA Receptors: A Northern Analysis Study".
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Gortner, G.; Pfenninger, M.; Kahl, G.; Weising, K. (1996). "Northern blot analysis of simple repetitive sequence transcription in plants".
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414:"Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes"
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Analysis of gene expression can be done by several different methods including RT-PCR, RNase protection assays, microarrays,
867:"Chronic cardiac rejection: Identification of five upregulated genes in transplanted hearts by differential mRNA display"
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Schlamp, K.; Weinmann, A.; Krupp, M.; Maass, T.; Galle, P. R.; Teufel, A. (2008). "BlotBase: A northern blot database".
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RNA run on a formaldehyde agarose gel to highlight the 28S (top band) and 18S (lower band) ribosomal subunits.
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Liang, P. Pardee, A. B. (1995) Recent advances in differential display. Current
Opinion Immunol. 7: 274β280.
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Capillary blotting system setup for the transfer of RNA from an electrophoresis gel to a blotting membrane.
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Bor, Y.C.; Swartz, J.; Li, Y.; Coyle, J.; Rekosh, D.; Hammarskjold, Marie-Louise (2006).
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65:, as well as in abnormal or diseased conditions. Northern blotting involves the use of
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Gilbert, S. F. (2000) Developmental
Biology, 6th Ed. Sunderland MA, Sinauer Associates.
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Yang, H.; McLeese, J.; Weisbart, M.; Dionne, J.-L.; Lemaire, I.; Aubin, R. A. (1993).
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Flow diagram outlining the general procedure for RNA detection by northern blotting.
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Yamanaka, S.; Poksay, K. S.; Arnold, K. S.; Innerarity, T. L. (1997).
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Trayhurn, P. (1996) Northern Blotting. Pro. Nutrition Soc. 55:583β589.
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589:"Simplified high throughput protocol for Northern hybridization"
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to separate RNA samples by size, and detection with a
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90:. The major difference is that RNA, rather than
74:technique was developed in 1977 by James Alwine,
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1109:(lipid:post translational modification)
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42:, or RNA blot, is a technique used in
412:Alwine JC, Kemp DJ, Stark GR (1977).
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1095:Electrophoretic mobility shift assay
94:, is analyzed in the northern blot.
563:10.1111/j.1432-1033.1991.tb15915.x
517:10.1111/j.1471-4159.1993.tb07465.x
106:to isolate only those RNAs with a
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1074:(post translational modification)
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201:) for which the ligand (e.g.,
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1277:Molecular biology techniques
994:Resources in other libraries
418:Proc. Natl. Acad. Sci. U.S.A
222:Advantages and disadvantages
27:Molecular biology technique
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871:Proc. Natl. Acad. Sci. USA
373:10.1016/j.gene.2008.08.026
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1198:Site-directed mutagenesis
989:Resources in your library
268:gene expression profiling
171:gel electrophoresis with
892:10.1073/pnas.91.14.6463
770:10.1002/elps.1150170702
439:10.1073/pnas.74.12.5350
1158:: key methods of study
941:10.1006/geno.2000.6427
829:10.1006/abio.1993.1189
673:10.1038/nprot.2008.216
605:10.1093/nar/21.14.3337
593:Nucleic Acids Research
486:10.1038/nprot.2006.216
195:horseradish peroxidase
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86:, named for biologist
78:, and George Stark at
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256:Reverse northern blot
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721:10.1101/gad.11.3.321
305:Differential display
247:acetylcholinesterase
236:diethylpyrocarbonate
191:alkaline phosphatase
1173:Gel electrophoresis
883:1994PNAS...91.6463U
430:1977PNAS...74.5350A
80:Stanford University
71:hybridization probe
1193:Restriction digest
1156:Molecular genetics
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1103:(protein:protein)
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975:Library resources
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290:Northwestern blot
187:chemiluminescence
44:molecular biology
18:Northern blotting
16:(Redirected from
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129:densitometry
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108:poly(A) tail
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1004:OpenWetWare
133:microarrays
1226:Microarray
311:References
76:David Kemp
1068:(protein)
708:Genes Dev
189:in which
124:formamide
98:Procedure
1271:Category
1255:Category
949:11161795
929:Genomics
786:36857667
689:24980302
681:19131955
533:33955961
381:18838116
274:See also
1047:General
911:8022806
879:Bibcode
837:7685563
778:8855401
730:9030685
623:8341618
571:1709098
525:8245974
426:Bibcode
228:RNA-Seq
157:agarose
977:about
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899:
835:
784:
776:
728:
687:
679:
621:
614:309787
611:
569:
531:
523:
458:414220
456:
449:431715
446:
379:
203:avidin
199:biotin
180:Probes
137:RT-PCR
1062:(RNA)
1056:(DNA)
902:44222
782:S2CID
743:e175.
685:S2CID
529:S2CID
945:PMID
907:PMID
833:PMID
774:PMID
726:PMID
677:PMID
619:PMID
567:PMID
521:PMID
454:PMID
377:PMID
361:Gene
250:mRNA
173:urea
143:Gels
56:mRNA
38:The
937:doi
897:PMC
887:doi
825:doi
821:210
766:doi
716:doi
669:doi
609:PMC
601:doi
559:doi
555:197
513:doi
481:doi
444:PMC
434:doi
369:doi
365:427
205:or
193:or
135:or
92:DNA
52:RNA
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943:.
933:71
931:.
919:^
905:.
895:.
885:.
875:91
873:.
869:.
857:^
845:^
831:.
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780:.
772:.
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760:.
748:^
724:.
712:11
710:.
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683:.
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663:.
631:^
617:.
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597:21
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375:.
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351:^
328:^
139:.
1148:e
1141:t
1134:v
1032:e
1025:t
1018:v
951:.
939::
913:.
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839:.
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20:)
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