RNA amplification and transcriptional profiling for analysis of stem cell populations.
As stem cells constitute a rare cell pool, a global analysis of gene expression is typically not possible. Here, we use a T7-based RNA amplification method combined with high-density oligonucleotide array technology to analyze gene expression. We isolated RNA from ES cells (100-100,000 cells), subjected them to two rounds of amplification, and compared each sample using microarray analysis. RNA isolation and amplification was highly reproducible and sensitive for the analysis of low abundant transcripts. Greater than 93% of the transcripts expressed were changed less than 2-fold when comparing the results of amplified RNA (100 cells to 100,000 cells) to unamplified RNA (isolated from 1,000,000 cells). This transcriptional analysis resulted in minimal skewing of gene expression. Using this technology, we analyzed the genetic programs of ES cells, STO feeder cells, and adult cardiomyocytes. We conclude that these technologies can be applied to the analysis of the genetic programs of rare cell populations and ultimately this analysis will enhance our understanding of the regulatory mechanisms of stem cell populations.
Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA.
Teresa D
TD
Gallardo
Robert E
RE
Hammer
Daniel J
DJ
Garry
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D008297:Male; D051379:Mice; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000302:isolation & purification; D015203:Reproducibility of Results; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
D012313:RNA