Genome-wide transcriptome analysis of 150 cell samples.
A major challenge in molecular biology is interrogating the human transcriptome on a genome wide scale when only a limited amount of biological sample is available for analysis. Current methodologies using microarray technologies for simultaneously monitoring mRNA transcription levels require nanogram amounts of total RNA. To overcome the sample size limitation of current technologies, we have developed a method to probe the global gene expression in biological samples as small as 150 cells, or the equivalent of approximately 300 pg total RNA. The new method employs microfluidic devices for the purification of total RNA from mammalian cells and ultra-sensitive whole transcriptome amplification techniques. We verified that the RNA integrity is preserved through the isolation process, accomplished highly reproducible whole transcriptome analysis, and established high correlation between repeated isolations of 150 cells and the same cell culture sample. We validated the technology by demonstrating that the combined microfluidic and amplification protocol is capable of identifying biological pathways perturbed by stimulation, which are consistent with the information recognized in bulk-isolated samples.
Irimia
Daniel
D
BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospital for Children, and Harvard Medical School, Boston, MA 02114, USA.
Mindrinos
Michael
M
Stanford Genome Technology Center and Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Russom
Aman
A
BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospital for Children, and Harvard Medical School, Boston, MA 02114, USA.
Xiao
Wenzhong
W
Stanford Genome Technology Center and Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Wilhelmy
Julie
J
Stanford Genome Technology Center and Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Wang
Shenglong
S
NuGEN Technologies Inc., San Carlos, CA 94070, USA.
Heath
Joe Don
JD
NuGEN Technologies Inc., San Carlos, CA 94070, USA.
Kurn
Nurith
N
NuGEN Technologies Inc., San Carlos, CA 94070, USA.
Tompkins
Ronald G
RG
BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospital for Children, and Harvard Medical School, Boston, MA 02114, USA.
Davis
Ronald W
RW
Stanford Genome Technology Center and Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Toner
Mehmet
M
BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Hospital for Children, and Harvard Medical School, Boston, MA 02114, USA.
D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D002874:Chromosome Mapping / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation* / Q000379:methods; D012313:RNA / Q000235:genetics* / Q000302:isolation & purification
D012313:RNA