Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling.
Laser capture microdissection (LCM) is used extensively for genome and transcriptome profiling. Traditionally, however, DNA and RNA are purified from separate populations of LCM-harvested cells, limiting the strength of inferences about the relationship between gene expression and gene sequence variation. There have been no published protocols for the simultaneous isolation of DNA and RNA from the same cells that are obtained by LCM of patient tissue specimens. Here we report an adaptation of the Qiagen AllPrep method that allows the purification of DNA and RNA from the same LCM-harvested cells. We compared DNA and RNA purified by the QIAamp DNA Micro kit and the PicoPure RNA Isolation kit, respectively, from LCM-collected cells from adjacent tissue sections of the same specimen. The adapted method yields 90% of DNA and 38% of RNA compared with the individual methods. When tested with the GeneChip 250K Nsp Array, the concordance rate of the single nucleotide polymorphism heterozygosity calls was 98%. When tested with the GeneChip U133 Plus 2.0 Array, the correlation coefficient of the raw gene expression was 97%. Thus, we developed a method to obtain both DNA and RNA material from a single population of LCM-harvested cells and herein discuss the strengths and limitations of this methodology.
Xu
Chang
C
Department of Otolaryngology-Head and Neck Surgery, University of Washington, Seattle, WA, USA.
Houck
John R
JR
Fan
Wenhong
W
Wang
Pei
P
Chen
Yu
Y
Upton
Melissa
M
Futran
Neal D
ND
Schwartz
Stephen M
SM
Zhao
Lue P
LP
Chen
Chu
C
Mendez
Eduardo
E
D004273:DNA, Neoplasm / Q000302:isolation & purification*; D020869:Gene Expression Profiling; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011786:Quality Control; D012334:RNA, Neoplasm / Q000302:isolation & purification*
D004273:DNA, Neoplasm; D012334:RNA, Neoplasm