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[DESCRIPTION]
<p>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 tRNAs<sup>1</sup>. 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 tRNAs<sup>2</sup>. 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. </p>
<p> </p>
<p>References</p>
<p>1. Dittmar KA, Goodenbour JM, Pan T. Tissue-specific differences in human transfer RNA expression. <em>PLoS Genetics</em>. 2006, 2(12):e221. DOI: <a href="https://doi.org/10.1371/journal.pgen.0020221" target="_blank">10.1371/journal.pgen.0020221</a> </p>
<p>2. Dittmar KA, Mobley EM, Radek AJ, Pan T. Exploring the Regulation of tRNA Distribution on the Genomic Scale. <em>Journal of Molecular Biology</em>. 2004, 337:31–47. DOI: <a href="https://doi.org/10.1016/j.jmb.2004.01.024" target="_blank">10.1016/j.jmb.2004.01.024</a></p>
<p>3. Kirchner <em>et al</em>. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. <em>PLoS Biology. </em>2017, <em>in press.</em></p>
<p>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. <em>PLoS Genetics</em>. 2013, 9(8):e1003767. DOI: <a href="http://dx.doi.org/10.1371/journal.pgen.1003767" target="_blank">http://dx.doi.org/10.1371/journal.pgen.1003767</a></p>

[BEFORE_START]
<p>This assay is based on a custom-made microarray not yet comercially available. Arrays are printed onto (3-glycidyloxypropyl)trimethoxysilane (Sigma) activated glas slides using a contact-spotter (QArray 2, Moleculare Devices). tRNA probes are arrayed in 384 well plates with a final concentration of 10 µM in 3× SSC/0.01% SDS in a total volume of 25 µl. After spotting, microarrays are incubated overnight at 4°C in a humidified atmosphere and subsequently dried for 30 min at 30°C. Arrays should be stored in a dry atmosphere at room temperature until use. tDNA probes are spotted in blocks of 100, whereby each tDNA probe is present 2x in each block. Probes targeting three tRNA standards are represented 3x in each block. Each microarray itself consists of 12 blocks. Thus, each tDNA probe is in total spotted 24x and each tRNA standard probe is spotted 36x on each array, respectively. Sequences of tDNA probes targeting the human tRNAome can be found in the attached Excel file. For more information on the microarray design or information on microarrays available for other organisms (mouse, <em>E. coli</em>, <em>B. subtilis</em>, etc.) please contact Prof. Zoya Ignatova (University of Hamburg, Hamburg, Germany) directly.</p>

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