Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.
Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.
Erickson
Heidi S
HS
Pathogenetics Unit, Laboratory of Pathology and Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. HSE@nig.gov
Albert
Paul S
PS
Gillespie
John W
JW
Wallis
Benjamin S
BS
Rodriguez-Canales
Jaime
J
Linehan
W Marston
WM
Gonzalez
Sergio
S
Velasco
Alfredo
A
Chuaqui
Rodrigo F
RF
Emmert-Buck
Michael R
MR
D020869:Gene Expression Profiling / Q000379:methods*; D006651:Histocytochemistry; D006801:Humans; D008297:Male; D042282:Microdissection / Q000379:methods*; D011471:Prostatic Neoplasms / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger; D012334:RNA, Neoplasm