Novel multiple 5'-amino-modified primer for DNA microarrays.
For DNA microarray analysis, total RNA is reverse-transcribed, labeled by incorporating fluorescent dye into the cDNA, and used to hybridize microarray. This protocol requires a minimum of 20 microg of total RNA. To overcome the sample limitation, an RNA amplification technique has been developed. Although it needs less RNA, this amplification technique is relatively expensive, time consuming, and, unfortunately, has been found to introduce bias. In this study, we designed a novel 5'-amino-modified primer and used it for priming cDNA synthesis. The novel primer has a special structure that contains four Uni-Link molecules with two nucleotide (thymine) residues inserted between them as spacers. This novel primer is used in the reverse-transcription reaction for cDNA synthesis. Using the novel 5'-modified primer combined with indirect labeling method, cDNA probes can be prepared with much less total RNA (5 microg or less) without amplification producing optimal results after hybridization of arrays. This primer can also be used to label nucleotides for other purposes.
Han
Jing
J
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Bethesda, MD 20892, USA.
Lee
Hin
H
Nguyen
Nga Yen
NY
Beaucage
Serge L
SL
Puri
Raj K
RK
D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D005821:Genetic Techniques; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012313:RNA