Technical Note pubs.acs.org/ac Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis Anita Rogacs, Yatian Qu, and Juan G. Santiago* Department of Mechanical Engineering, Stanford University, Stanford, California, 94305 *S Supporting Information ABSTRACT: We demonstrate a novel assay for physico- chemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida. This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR. ucleic acid amplification methods, including reverse magnetic beads. These approaches are well-established in N transcription-quantitative polymerase chain reaction traditional settings but require multiple reagent wash steps and (RT-qPCR), targeting the 16S rRNA (16S rRNA) enable fast specialized fabrication or manipulation (e.g., pumping liquids and specific detection of bacteria in complex samples such as for wash steps or moving magnets for beads). Further, SPE whole blood.1,2 16S rRNA is a universal constituent of bacterial binding capacity can be low due to competitive protein ribosomes present at high copy numbers (103−104 per actively absorptions (e.g., to silica) and the presence of PCR inhibiting 3,4 growing cell). Targeting these biomarkers can potentially chemistry such as guanidinium thiocyanate (GuCN), guanidi- increase assay sensitivity compared with the assays targeting the nium chloride (GuHCl), isopropyl alcohol, or ethanol. 5,6 corresponding DNA. Sample preparation is time-consuming Of the few studies reporting RNA purification using a − and labor-intensive, involving one or more centrifugation steps, microfluidic platform,20 27 to our knowledge, only two have ff bu er exchange(s), lysing, and associated multistep processes of reported using blood or blood product as a sample matrix.20,22 fi nucleic acid extraction and puri cation from PCR inhibitors. Witek et al. employed an array of photoactivated polycarbonate The latter can include endogenous species, such as heme micropillars as the solid phase to purify total RNA from proteins, lactoferrin, and immunoglobin, as well as species used bacterial cells suspended in whole blood.22 Their protocol in preparation, including heparin, denaturants, surfactants, 7,8 eliminated the PCR-inhibiting guanidine chemistry by adopting chaotropic salts, and alcohols. These sample preparation thermal and mechanical lysing approaches. They also report a steps can be difficult to integrate, automate, or hasten, 9,10 high, postextraction RNA integrity. However, their assay did particularly in a miniaturized system. not address the need for adequate RNase control4,22 (see Tsui Fundamentally, RNA can be an exceedingly challenging et. al11 for a discussion of RNA stability in blood products). We blood biomarker due to its extreme lability. RNA is susceptible attribute their high RNA integrity to the very large number of to the ubiquitous action of ribonucleases (RNases)11 and bacteria they spiked into their blood sample. We estimate they degradation to physicochemical conditions including elevated ° 12 13−15 spiked 18 000 ¢/nL-blood (where ¢ is the number of bacterial temperature (>65 C) and high pH. Careful decontami- 6 nation together with assay standardization or automation can cells), which is on the order 10 -fold higher bacteria ff concentration than the current work. Further, their protocol mitigate the e ects of exogenous RNase, but only careful design ffi of assay chemistry can provide sufficient protection against the required ethanol, which can inhibit PCR if not su ciently removed. abundant endogenous RNases in blood. As one salient example, 20 without adequate RNase control, free RNA is nonamplifiable Instead of SPE, Root et al. used an oligonucleotide polymer 11 capture matrix to purify free RNA spiked into serum. This after 15 s of incubation in plasma or serum. fi To date, the majority of microfluidic-based sample aqueous puri cation process reported an impressive 375 RNA preparation approaches have focused on DNA isolation, and mainly via the miniaturization of solid-phase extraction (SPE) Received: April 17, 2012 − methods.16 19 SPE-type approaches include specialized struc- Accepted: June 12, 2012 tures such as packed beads, monolithic porous structures, and © XXXX American Chemical Society A dx.doi.org/10.1021/ac301021d | Anal. Chem. XXXX, XXX, XXX−XXX Analytical Chemistry Technical Note Figure 1. Schematic summarizing protocol with one mixing and two dispensing steps for otherwise automated on-chip RNA extraction from whole blood. TE mixed with lysate (brown) contains target nucleic acid (green), proteins, and potential PCR-inhibiting chemistries. Appropriate selection of trailing and leading ions enables selective focusing of target nucleic acid while leaving PCR inhibitors behind. The detail view shows on-chip extraction of RNA from blood stained with SYBR Green II, focused into a concentrated zone. The amplification plot shows the result of alkaline- based lysing (enhanced with Triton X-100, DTT, and carrier RNA) of total nucleic acid from whole blood spiked with P. putida at 30 ¢/nL, followed by purification of total nucleic acid (NA) from lysate using ITP. The NA collected from the output well was split to perform both RT-qPCR and qPCR to verify successful extraction of 16S rRNA (red dotted) and 16S rDNA (red bold dashed). For the negative control template (uninfected blood), RT-qPCR amplified 16S rRNA (blue solid line) above 30 cycles and qPCR did not amplify 16S rDNA within 40 cycles, as expected. copies/μL of serum.20 However, very importantly, the Root up to 1,000,000-fold preconcentration.32 We have demon- assay included no lysing strategy; instead, they demonstrated strated successful ITP extraction of small RNA from cell culture purification by spiking free RNA into a preprepared serum lysate,33 micro-RNA from total RNA,34,35 genomic DNA sample that already contained 0.5% w/v concentration of (gDNA)36 and pathogenic DNA (malaria)37 from whole lithium dodecyl sulfate, a powerful RNase inhibitor. blood lysate, and rRNA from bacteria in urine lysate.38 We know of no reports of miniaturized systems that can However, no previous nucleic acid extraction protocols using extract RNA from blood or blood lysate at RNA copy numbers ITP have been designed for adequate RNase control and RNA which are within a factor of about 105 orders of magnitude of integrity. clinically relevant RNA levels. For that matter, we know of no In this study, we offer new lysing and ITP chemistry that can studies that have combined lysing and RNase control into a isolate total nucleic acid from Gram-negative bacteria single chemistry. There is, therefore, still a significant need for a suspended in whole blood. We demonstrate integration of microfluidic assay that has adequate RNase control and can our method with qPCR, one of the most common transduction enable direct integration of lysis and nucleic acid purification assays in molecular biology and an assay with well-characterized ff (particularly RNA) and also provide nucleic acid samples sensitivity to impurities and bu er conditions. Our protocol is compatible with amplification methods. unique in that it provides a combined lysis and TE chemistry Isotachophoresis (ITP) offers an alternative approach to that protects RNA from both exogenous and endogenous fi nucleic acid extraction and purification, and the input and RNase degradation during extraction and ITP-based puri ca- tion. This aspect is critical for RNA isolation from RNase-rich output reagents used in ITP can be compatible with lysis, fi RNase control, and amplification. ITP does not require specific matrices, such as whole blood. Although the puri ed extract can surfaces, specialized geometries, or pumping of reagents. It uses be used to target a wide range of DNA and RNA from whole an electric field to extract and preconcentrate only target blood, we here target 16S rRNA to demonstrate assay analytes whose electrophoretic mobility is bracketed between compatibility with RNA and to highlight the enhanced the anions of its trailing (TE) and leading electrolytes (LE). sensitivity achievable by targeting highly transcribed genes. Anionic inhibitors with mobilites lower than of the TE do not focus, but do electrophorese into the microchannel. The ■ MATERIALS AND METHODS separation distance between these potentially PCR-inhibiting A schematic of our extraction process is shown in Figure 1. We contaminants and the focused nucleic acid in the ITP zone started with human whole blood infected with known increases over time. For a channel length, L, the separation concentrations of Pseudomonas putida cells. We chemically distance between the ITP and the inhibitor zone front, ΔL, can lysed the blood at room temperature for 1 min in a mixture of Δ − μ μ μ μ be expressed as L =(1 i/ TE)L, where i and TE are the sodium hydroxide (NaOH) (pH > ∼12), dithiothreitol (DTT) inhibitor and TE anion mobilities, respectively. For example, reducing agent, Triton X-100 nonionic surfactant, and synthetic μ μ the zone front of an inhibitor with mobility i = 0.9 TE will lag carrier RNA. We then “quenched” this high pH with ice-cold 0.6 cm behind the ITP zone at the end of our 6 cm TE buffer and then directly pipetted this combined lysate and microchannel. ITP is a highly sensitive,28,29 robust30,31 sample TE into the input well of a microfluidic chip with a single preparation method that can, under ideal conditions, provide connecting channel (and single output well) prefilled with LE. B dx.doi.org/10.1021/ac301021d | Anal.