This application note describes the application of the Low RNA Input Fluorescent Linear Amplification kit for the use of poly (A)+ RNA or total RNA samples. It shows that micrograms of cRNA targets can be generated within 6 hours, using as little as 10 ng of poly (A)+ RNA, or 50 ng of total RNA sample input. The resulting cRNA targets were of high quality and per- formed reproducibly and robustly on Agilent's oligonu- cleotide microarrays. Furthermore, the log ratios of array results from poly (A)+ RNA sample input correlated with that from total RNA input. The easy-to-use Low RNA Input Fluorescent Linear Amplification kit can be applied to a wide range of samples for gene expression analysis.
Agilent Technologies developed the Low RNA Input Fluorescent Linear Amplification (LRIFLA) kit to address the challenge of using limited biological samples, such as rare primary cells or biopsy tissue, in DNA microarray analysis. The kit utilizes T7 RNA polymerase for a single round of linear amplification to produce sufficient quantities of fluorescently labeled cRNA or cDNA targets from as little as 10 ng of poly(A)* RNA or 50 ng of total RNA.
The document details experiments using HeLa and human spleen samples to demonstrate the kit's performance. Results show that micrograms of high-quality, labeled cRNA can be generated within six hours. The study confirms that the log ratios from arrays using amplified targets correlate well with those from standard total RNA inputs, validating the method for gene expression studies.
The kit can produce high-quality cRNA targets using as little as 10 ng of poly (A)* RNA.
The procedure supports using up to 5000 ng of total RNA sample input.
Micrograms of cRNA targets can be generated within 6 hours.