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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Rio, Donald C. |
| Abstract | This protocol is used to denature and separate large mRNA molecules (0.5-10 kb) on agarose gels by electrophoretic size fractionation. Glyoxal (also called diformyl or ethanedial), the agent responsible for maintaining denaturation in this protocol, contains two carbonyl groups that react to form a cyclic ring structure with the imino and amino groups of guanine. It can also react with the amino groups of adenine and cytidine. When RNA is denatured in the presence of glyoxal, this covalent adduct prevents normal base pairing and maintains the RNA in a denatured state in agarose gels. Once formed, these adducts are stable at room temperature at pH <7.0; thus, there is no need to add glyoxal to the gel or to the gel buffers to maintain the RNA in the denatured state. Because the fully denatured RNA migrates through agarose gels according to its molecular mass, this method can be used to accurately size mRNA molecules. Following electrophoresis and reversal of glyoxalation, the RNA can be detected using a northern hybridization procedure. |
| File Format | HTM / HTML |
| ISSN | 19403402 |
| Issue Number | 2 |
| Journal | Cold Spring Harbor Protocols |
| Volume Number | 2015 |
| e-ISSN | 15596095 |
| Language | English |
| Publisher | Cold Spring Harbor Laboratory Press |
| Publisher Date | 2015-02-02 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Clinical Laboratory Techniques Glyoxal Chemistry Nucleic Acid Denaturation Rna Base Pairing Electrophoresis, Agar Gel Journal Article |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry, Genetics and Molecular Biology |
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