The technique provides a method for performing gene expression analysis from a small amount of total RNA, including transcribing total RNA into cDNA, amplifying the cDNA by a linear amplification method to obtain sufficient amounts of Cy-labeled cRNA, and then performing microarray analysis on the Cy-labeled cRNA. The method requires only 10 nanograms of total RNA and takes less than 6.5 hours to complete.
Agilent Technologies has developed the Low Input Quick Amp (LIGA) Labeling Kit to address the need for gene expression profiling from increasingly smaller amounts of total RNA. The kit is based on a single-tube procedure employing AffinityScript Reverse Transcriptase, a mutant MMLV-RT that binds primer-template complexes with 10-fold higher efficiency than wild type. This results in increased cDNA yields and improved sensitivity, allowing the generation of Cy-labeled cRNA suitable for oligonucleotide microarray experiments with only 10 nanograms of total RNA input in less than 6.5 hours.
| Total RNA Input Requirement | 10 nanograms |
|---|---|
| Time to Data | less than 6.5 hours |
| Product Part Numbers | 5190-2305, 5190-2306 |
| Dynamic Range | ~ 5 logs |
The assay requires only 10 nanograms total RNA input.
The procedure takes less than 6.5 hours to generate sufficient quantities of Cy-labeled cRNA.
The kit employs AffinityScript Reverse Transcriptase, a mutant MMLV-RT.