Amine-modified random primers to label probes for DNA microarrays.
DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.
Xiang
Charlie C
CC
Laboratory of Genetics, National Institute of Mental Health, Bethesda, MD 20892, USA.
Kozhich
Olga A
OA
Chen
Mei
M
Inman
Jason M
JM
Phan
Quang N
QN
Chen
Yidong
Y
Brownstein
Michael J
MJ
D016475:3T3 Cells; D000818:Animals; D017931:DNA Primers / Q000032:analysis* / Q000235:genetics; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000032:analysis*; D015870:Gene Expression; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results
D017931:DNA Primers; D005456:Fluorescent Dyes; D012313:RNA