Simultaneous isolation of DNA and RNA from the same cell population obtained by laser capture microdissection for genome and transcriptome profiling

Abstract

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.

Figure 1

A representative tissue specimen quality assessment result. After OCT embedment, the tissue was collected by scraping from slides after sectioning (A), after storage at −80°C for 48 hours (B), after HistoGene staining (C), and after LCM (D); tissue sectioning and staining were performed as previously described. RNA was extracted from the scraped tissues, and the integrity of the purified RNA was assessed by an Agilent 2100 bioanalyzer. The x axis indicates the relative migration or size of the nucleic acid and the y axis displays the fluorescence or quantity. At right is the virtual gel of the sample generated by the bioanalyzer. RIN, RNA integrity number.

Figure 2

Comparison of different extraction conditions. LCM-harvested cells were incubated with AllPrep RLT Plus extraction buffer at room temperature for 4 hours (lane 1: RNA; lane 6: DNA); at room temperature overnight (lane 2: RNA; lane 7: DNA); at 42°C overnight (lane 3: RNA; lane 8: DNA); and at 55°C overnight (lane 4: RNA; lane 9: DNA). Purified DNA and RNA were revealed on a 1% agarose gel. Lane 5 shows the 1-kb DNA size ladder.

Figure 3

Using the DNA purified by the adapted AllPrep DNA/RNA method for genome profiling. A: Amplified tumor DNA fragments from DNA purified by the adapted AllPrep DNA/RNA method and by the QIAamp DNA purification method as revealed on a 2% agarose gel. Each DNA sample was amplified in three independent polymerase chain reaction amplifications as shown in three lanes. DirectLoad wide range DNA marker was used on both sides as size ladder. B: Amplified tumor DNA fragments from DNA purified by the TRIzol method and by the QIAamp DNA purification method as revealed on a 2% agarose gel. Size marker is the same as in A. C: Whole genome CNV measured on the Affymetrix GeneChip Human Mapping 250K Nsp Array using DNA purified by the adapted AllPrep DNA/RNA method (AllPrep) and by the QIAamp DNA purification method (QIAamp). CNV of buffy coat DNA from each patient (1–3) was also presented as reference. x axis, SNP locations; y axis, log_2 signal intensity ratios; the gray horizontal line is for y = 0. D: Details of CNV on chromosome 11 (left) and chromosome 20 (right).

Figure 4

Using the RNA purified by the adapted AllPrep DNA/RNA method for expression profiling. A: Representative spectra of cRNA after two-round linear amplification, as revealed by the Agilent 2100 bioanalyzer from RNA purified by the adapted AllPrep DNA/RNA method (middle) and by the PicoPure method (right). Left panel shows the RNA size ladder. B: Scatter plots showing the correlation of gene expression measured on Affymetrix GeneChip HG-U133 Plus 2.0 Array using RNA purified by the adapted AllPrep DNA/RNA method (y axis) and by the PicoPure method (x axis). The Pearson correlation coefficient (r) for each test pair is as presented.