This document introduces a method for optimizing cRNA fragmentation using the Agilent 2100 bioanalyzer for microarray experiments.
This document outlines a workflow for creating fluorescence-labeled cRNA from HeLa RNA and optimizing its fragmentation for microarray hybridization. It highlights the Agilent 2100 bioanalyzer as a faster and more sensitive alternative to gel electrophoresis for monitoring sample quality and fragmentation size.
The text details the use of the RNA 6000 LabChip kit to analyze total RNA, mRNA, and cRNA samples. It explains how to assess RNA quality by checking for ribosomal RNA contamination and how to determine the optimal fragmentation time for cRNA to ensure a majority of fragments fall within the 50-200 base pair range required for optimal microarray performance.
| Analysis time | about 25 minutes |
|---|---|
| Sample volume | 25 ng |
| Optimal fragment size | 50-200 bases |
| Fragmentation buffer | 10 mM zinc acetate |
The bioanalyzer is faster and more sensitive than gel electrophoresis.
The cRNA should be fragmented to an optimal size of 50-200 bases long.
The bioanalyzer requires only 25 ng of RNA sample.
A failed reaction is indicated if the free dye peak is more than two-fold higher than the cRNA sample peak.