Selective nucleic acid removal via exclusion (SNARE): capturing mRNA and DNA from a single sample

Abstract

The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method.

Figure 1

A) (left) Picture of SNARE device with dimensions labeled and (right) top down schematic of SNARE device with wells labeled. Note the two fluid paths. One on the front of the device and one on the back. mRNA extraction occurs along the front and DNA extraction occurs along the back. B) Operation of SNARE for mRNA and DNA extraction and purification from a single sample. Steps 1–3 show front side of SNARE for mRNA isolation. Steps 4–6 show backside of SNARE for DNA isolation. (PMPs: Paramagnetic Particles)

Figure 2

RNA and DNA extraction methods from a single sample, using the traditional Trizol (guanidinium thiocyanate-phenol-chloroform) or Spin Column methods as compared to SNARE

Figure 3

Relative mRNA and DNA GAPDH signal isolated using SNARE for the comparison of different lysis/mRNA binding buffers. Based on this data, Modified LIDS was recommended for use in SNARE *p<0.03, ** p<0.001, + p<0.001, ++ p<0.04 Sample size per a group n=6.

Figure 4

Comparison of A) relative GAPDH mRNA, and B) GAPDH DNA signal purified from 1000, 100, 10 or 1 LNCaPs using SNARE (grey dots) or Qiagen (black dots). Each dot represents a nucleic acid purification procedure with horizontal lines representing the mean of the individual experiments. Sample size per a group n=6.

Figure 5

A) Sequencing chromatogram and alignment of exon 5 and 6 of the AR from mRNA purified from 10 LNCaP cells using the SNARE method. B) Sequencing chromatogram and alignment of exon 8 of the AR from DNA purified from 10 LNCaPs using the SNARE method. The T887A LNCaP mutation was identified (black box).