RNA expression profiling at the single molecule level.
We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.
Hesse
Jan
J
Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria.
Jacak
Jaroslaw
J
Kasper
Maria
M
Regl
Gerhard
G
Eichberger
Thomas
T
Winklmayr
Martina
M
Aberger
Fritz
F
Sonnleitner
Max
M
Schlapak
Robert
R
Howorka
Stefan
S
Muresan
Leila
L
Frischauf
Anna-Maria
AM
Schütz
Gerhard J
GJ
D002460:Cell Line; D015342:DNA Probes; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D018076:DNA, Complementary