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Cell Biology JoVE (Open Access) Citable · DOI

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

DOI: 10.3791/2007-v
What you'll learn
  • ✓ Isolate total charged tRNA from S. cerevisiae cells using acid extraction
  • ✓ Label tRNA samples with Cy3/Cy5 fluorescent dyes for microarray detection
  • ✓ Perform genome-wide microarray hybridization to quantify aminoacylation levels
  • ✓ Analyze microarray data to determine relative tRNA charging across the genome
Protocol

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Difficulty
advanced
Total time
~6-8 hours per sample (including overnight hybridization)
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1

Steps

1
Isolate total charged tRNA from yeast cells

Extract total charged tRNA from S. cerevisiae using acid precipitation and purification methods to preserve aminoacylation status. This step maintains the native charged state of tRNAs before analysis.

▶ 00:45
2
Label tRNA samples with Cy3 and Cy5 dyes

Apply fluorescent Cy3 and Cy5 labeling to charged and control tRNA samples respectively, enabling dual-channel microarray detection and comparison of aminoacylation levels.

▶ 02:37
3
Hybridize labeled tRNA to microarray slides

Perform microarray hybridization with labeled tRNA samples and conduct image scanning and data acquisition to obtain fluorescent signals for all tRNA species.

▶ 05:15
4
Analyze microarray results and aminoacylation data

Process microarray images and calculate relative aminoacylation levels for all yeast tRNAs by comparing Cy3/Cy5 signal ratios across the genome.

▶ 06:33
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