The stage-specific testicular germ cell apoptotic response to low-dose X-irradiation and 2,5-hexanedione combined exposure. I: Validation of the laser capture microdissection method for qRT-PCR array application.
Over the past decade, laser capture microdissection (LCM) has grown as a tool for gene expression profiling of small numbers of cells from tumor samples and of specific cell populations in complex tissues. LCM can be used to study toxicant effects on selected cell populations within the testis at different stages of spermatogenesis. There are several LCM-related hurdles to overcome, including issues inherent to the method itself, as well as biases that result from amplifying the LCM-isolated RNA. Many technical issues associated with the LCM method are addressed here, including increasing RNA yield and obtaining more accurate quantification of RNA yields. We optimized the LCM method optimized to generate RNA quantities sufficient for quantitative reverse transcription polymerase chain reaction (qRT-PCR) array analysis without amplification and were able to validate the method through direct comparison of results from unamplified and amplified RNA from individual samples. The addition of an amplification step for gene expression studies using LCM RNA resulted in a bias, especially for low abundance transcripts. Although the amplification bias was consistent across samples, researchers should use caution when comparing results generated from amplified and unamplified LCM RNA. Here, we have validated the use of LCM-derived RNA with the qRT-PCR array, improving our ability to investigate cell-type and stage-specific responses to toxicant exposures.
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, USA.
Natasha R
NR
Catlin
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, USA.
Susan M
SM
Huse
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island, USA Kim_Boekelheide@brown.edu.
Kim
K
Boekelheide
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods*; D006588:Hexanones / Q000633:toxicity*; D061346:Laser Capture Microdissection / Q000379:methods*; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013091:Spermatogenesis / Q000187:drug effects / Q000528:radiation effects; D013737:Testis / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D014965:X-Rays
D006588:Hexanones; C011269:2,5-hexanedione
PCR array.; amplification; laser capture microdissection; testis