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Comparison of different methods and approaches for the analysis of human ES-cell gene expression |
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| Sperger et al. [1] |
Sato et al. [2] |
Richards et al. [3] |
Abeyta et al. [4] |
Zeng et al. [5] |
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| Human ES-cell lines used |
H1, H7, H9, H13, H14 |
H1 |
HES3, HES4 |
H9, HSF-1, HSF-6 |
BG01, BG02 |
| Culture conditions |
MEFs |
Matrigel |
MEFs |
MEFs |
MEFs |
| Method of ES-cell isolation |
Treatment with collagenase until colonies lifted off the MEFs |
Treatment with dispase until cells were free of MEFs |
Microdissection to free colonies of MEFs |
Mechanical dissection of colonies from MEFs, then collagenase treatment |
Trypsinization |
| Arrays used |
Stanford microarrays |
Affymetrix arrays (hU133A and mouse U74Av2) |
SAGE |
Affymetrix arrays (hU133A and hU133B) |
Custom 16,659-spot 70-bp oligonucleotide array |
| Cells compared |
hEC, hES and seminoma |
hES and published mES [16] |
hES and hES and additional SAGE libraries [24] |
hES and hES versus published mES [16] |
hES and hES versus mES [28] |
| Primary subtraction method |
Somatic and cancer cell lines |
Differentiated hES cells |
None |
None |
Pooled human RNA |
| Software/analysis used |
Significance analysis of microarrays (SAM) [25] |
dChip and MAS 4.0 (Affymetrix) |
Comparison of two SAGE resources with SAGE 2000 [26,27] |
MAS 5.0 (Affymetrix) |
Gene Pix (Axon Instruments) |
| Number of genes enriched in human ES cells |
1,760 |
918 |
8,341 |
7,385 |
373 |
| Candidate pluripotency genes* |
565 |
227 |
192 |
76 |
92 |
| Confirmation of gene expression using |
RT-PCR |
RT-PCR |
RT-PCR |
Quantitative RT-PCR |
RT-PCR |
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*Candidate pluripotency genes are defined as genes that are found only in all pluripotent cell lines examined in each study. Abbreviations: hEC, human embryonal carcinoma cells; hES, human embryonic stem cells; MEFs, mouse embryonic fibroblasts; mES, mouse embryonic stem cells; RT-PCR, reverse-transcriptase-coupled PCR; SAGE, serial analysis of gene expression. | |||||
Pyle et al. Genome Biology 2004 5:235 doi:10.1186/gb-2004-5-8-235 |
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