PCR-based quantification of amplified RNA from laser microdissected mouse liver samples.
New molecular methods such as quantitative reverse transcription-polymerase chain reaction (RT-PCR) and microarray gene expression analysis have been established recently to quantify gene expression in tissue samples. Such methods, although highly sensitive, require RNA quantities of at least several micrograms. These amounts are not available in many experiments concerning microdissected embryonic or regenerating structures. We combined laser-assisted tissue preparation, RNA amplification, and quantitative RT-PCR to estimate both accuracy and linearity of gene expression in small tissue samples. Our results show that mRNA isolated from laser-microdissected fetal liver tissue or regenerative nodules, which originated from EGFP-marked transplanted fetal cells, can be significantly increased with the amplification protocol. The quantitative expression ratio of the genes albumin and GAPDH was conserved after one and two rounds of amplification compared to nonamplified material. Furthermore, genes expressed at low levels such as the transcription factor C/EBPbeta become detectable after two rounds of amplification in small microdissected tissue samples.
Cantz
Tobias
T
Department of Gastroenterology, Hepatology and Endocrinology, Hanover Medical School Hanover, Carl-Neuberg-Strabetae 1, D-30625, Hanover, Germany.
Jochheim
Andrea
A
Cieslak
Alexandra
A
Hillemann
Tina
T
Scharf
Jennifer
J
Manns
Michael P
MP
Ott
Michael
M
D000418:Albumins / Q000235:genetics; D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D004210:Dissection; D005333:Fetus; D020869:Gene Expression Profiling / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D022781:Hepatocytes / Q000637:transplantation; D007834:Lasers; D008099:Liver / Q000502:physiology; D051379:Mice; D012313:RNA / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D000418:Albumins; D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases