| Popis: |
This protocol describes the microarray-based quantification of human tRNAs. The tRNA microarray consists of 40 full-length tDNA probes recognising all 54 nuclearly encoded human isoacceptor tRNAs1. The array has been shown to reliably distinguish tRNA isoacceptors with a sequence difference of more than 8 nucleotides. Deacetylated tRNAs are selectively labeled with a fluorescent oligonucleotide hairpin which is covalently attached to the single-stranded 3' NCCA overhang, a commen feature of all tRNAs2. For comparison, differently labeled tRNAs from two different conditions or cell/tissue types are labeled with the oligonucleotide hairpins with two different colors, mixed and hybridized onto the tRNA microarray. Ratios between the two different fluorescent signals are then used for relative comparison of tRNAs between the two samples. The assay has also be applied to other eukaryotic or prokaryotic organisms. For more information please contact Prof. Zoya Ignatova (University of Hamburg, Hamburg, Germany). However, this protocol only describes the quantification of human tRNAs. Example of the successful usage of the tRNA array can be found in Ref. 3 and 4. References 1. Dittmar KA,Goodenbour JM, Pan T. Tissue-specific differences in human transfer RNA expression. PLoS Genetics. 2006,2(12):e221. DOI: 10.1371/journal.pgen.0020221 2. Dittmar KA, Mobley EM, Radek AJ, Pan T. Exploring the Regulation of tRNA Distribution on the Genomic Scale. Journal of Molecular Biology. 2004, 337:31–47. DOI: 10.1016/j.jmb.2004.01.024 3. Kirchner et al. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. PLoS Biology. 2017, in press. 4.Czech A, Wende S, Mörl M, Pan T, Ignatova Z. Reversible and Rapid Transfer-RNA Deactivation as a Mechanism of Translational Repression in Stress. PLoS Genetics. 2013, 9(8):e1003767. DOI: http://dx.doi.org/10.1371/journal.pgen.1003767 |
