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B. Why is RNA amplification
necessary?
Glass microarray
analysis experiments typically require 5-20 g of total RNA
per slide for sample labeling and hybridization. Thus,
microarray-based gene expression analysis of very small
samples [laser capture microdissection (LCM), tissue biopsies,
or other clinical samples] is difficult due to the very low
amounts of total RNA recovered from the samples. Linear
amplification of RNA from small samples produces sufficient
quantities of RNA for sample labeling and hybridization. Since
the amplification technique is highly reproducible and
maintains representation of the gene expression in the
original sample, it is recommended for probe synthesis by most
manufacturers of commercially available microarrays.
C. How do direct and indirect labeling of
aRNA differ?
Direct
labeling is incorporation of modified NTPs into
amplification products during the IVT step of the
amplification process. To make aRNA that is labeled with
fluorescent dyes, a mixture of dye-modified and unmodified (or
unlabeled) nucleotides are typically used in order to obtain
an optimal ratio of dye-labeled to unlabeled nucleotide for
maximal fluorescence. Usually ~200-400 M of dye-labeled CTP
is used with 1-3 mM unlabeled NTPs. Biotin-modified
nucleotides are incorporated fairly well with T7 RNA
polymerase. Ambion recommends using UTP:biotin-UTP ratios of
1:1 to 3:1. In ge
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