Rapid and Selective RNA Analysis by Solid-Phase Microextraction

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Rapid and Selective RNA Analysis by Solid-Phase Microextraction Omprakash Nacham, Kevin D. Clark, Marcelino Varona, and Jared L. Anderson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02733 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Analytical Chemistry

Rapid and Selective RNA Analysis by Solid-Phase Microextraction

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Omprakash Nacham, Kevin D. Clark, Marcelino Varona, and Jared L. Anderson*

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Department of Chemistry, Iowa State University, Ames, IA 50011 USA

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Abstract In this study, a solid-phase microextraction (SPME) method was developed for the purification of messenger RNA (mRNA) from complex biological samples using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for quantification. The chemical composition of the polymeric ionic liquid (PIL) and a poly acrylate (PA) SPME sorbent coating was optimized to enhance the extraction performance. Of the studied SPME sorbent coatings, the PIL containing carboxylic acid moieties in the monomer and halide-based anions extracted the highest amount of mRNA from aqueous solutions, whereas the native PA fiber showed the lowest extraction efficiency. On the basis of RT-qPCR data, electrostatic interactions and an ion-exchange mechanism between the negatively charged phosphate backbone of RNA and the PIL cation framework were the major driving forces for mRNA extraction. The optimized PIL-based SPME method purified a high quantity of mRNA from crude yeast cell lysate compared to a phenol/chloroform extraction method. The reusability and robustness of PIL-based SPME for RNA analysis represents a significant advantage over conventional silica-based solid-phase RNA extraction kits. The selectivity of the SPME method toward mRNA was enhanced by functionalizing the PA sorbent with oligo dT20 using carbodiimide-based amide linker chemistry. The oligo dT20-modified PA sorbent coating demonstrated superior extraction performance than the native PA sorbent coating with quantification cycle (Cq) values 33.74 ± 0.24 and 39, respectively. The modified PA sorbent extracted sufficient mRNA from total RNA at concentrations as low as 5 ng µL-1 in aqueous solutions without the use of organic solvents and time-consuming multiple centrifugation steps that are required in traditional RNA extraction methods.

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Corresponding Author:

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Jared L. Anderson Department of Chemistry 1605 Gilman Hall Iowa State University Ames, IA 50011 Tel.: +1 515-294-8356 E-mail address: [email protected]

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ACS Paragon Plus Environment


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Introduction

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The analysis of ribonucleic acid (RNA) is an essential component of gene expression

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studies and disease prognosis in biomedical applications. Analytical techniques including

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Northern blotting,1 ribonuclease protection assays,2 and real-time reverse transcription

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quantitative polymerase chain reaction (RT-qPCR)3 are commonly employed for the detection

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and quantification of messenger RNA (mRNA). However, the co-extraction of proteins,

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phospholipids, or chemically similar DNA from biological sources results in poor method

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accuracy, irreproducible measurements, or may inhibit mRNA assays altogether.4 When

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combined with the complexity of detecting low abundance mRNA transcripts, the purification

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and preconcentration of mRNA represents a major bottleneck in the bioanalytical workflow.

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Traditional approaches to RNA purification involve liquid-liquid extraction (LLE) and

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rely on the differential partitioning of RNA and contaminants between an aqueous phase and

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organic layer. Although there are numerous variations of LLE that utilize surfactant additives,

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chaotropic salts, or enzymes to improve yields/purity of RNA extracts, the excessive use of

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organic solvents, time-consuming centrifugation procedures, and multiple sample transfer steps

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are significant drawbacks to this classical method. To decrease analysis time and reduce organic

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solvent consumption, solid phase extraction (SPE) methods were developed that rely on the

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reversible binding of RNA to a silica-based sorbent. The solid matrix can also be functionalized

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with oligo deoxythymine (dT) for the specific capture of mRNA from total RNA samples via

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hybridization with the naturally occurring 3’ polyadenylated tails of mRNA.5 However,

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commercial SPE kits rely on centrifugation or applied vacuum and are not recommended for re-

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use making this method difficult to automate and dramatically increase the cost of analyzing

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multiple samples.


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Solid-phase microextraction (SPME) is a solvent-free, non-exhaustive sample

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preparation technique that utilizes a thin layer of sorbent material immobilized on a solid support

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to extract analytes from a sample.6 SPME has been employed for the extraction and

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quantification of peptides,7 drug substances,8 and metabolites9 in vitro and in vivo to provide

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insight toward biological systems. The partitioning of analytes in SPME is inextricably related to

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the chemical composition of the sorbent material. Polymeric ionic liquids (PILs) represent a new

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class of SPME sorbent coatings with tunable solvation properties and high thermal stabilities.11

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Recently, PILs consisting of readily interchangeable cations/anions with polymerizable groups

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have recently been investigated as sorbents for the extraction of DNA.10-12

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In contrast to DNA, RNA is chemically less stable and highly susceptible to enzymatic

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degradation.13 In addition to purification challenges, RNA quantification is not straightforward

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and involves multi-step protocols (e.g., reverse transcription and qPCR) that increase the overall

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complexity of the analysis. Given the success of SPME toward DNA analysis, the development

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of SPME-based purification technologies for RNA would provide the following advantages over

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silica-based SPE kits: (1) the chemical composition of PIL-based sorbent phases can be readily

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customized to possibly enhance the RNA extraction performance; (2) electrostatic and ion-

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exchange interactions are the major driving forces for RNA extraction, minimizing the use of

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toxic organic solvents, chaotropic salts, and alcohols (known PCR inhibitors); (3) PIL sorbent

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coatings are resistant to harsh chemical conditions that would otherwise degrade contemporary

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silica-based sorbents and can be easily regenerated by exposing the PIL to high ionic strength

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solutions; (4) owing to its simplicity and portability, a SPME-based RNA purification platform

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would be highly suitable for field sampling and point-of-care diagnostic applications.



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To our knowledge, this study constitutes the first report in the development and

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application of a SPME approach that is coupled to RT-qPCR for selective mRNA analysis from

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crude yeast cell lysate. The chemical composition of the PIL and polyacrylate SPME sorbent

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coatings were optimized for efficient mRNA extraction. Under similar experimental conditions,

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the PIL sorbent phase comprised of polar functional groups (carboxylic acid) within the IL

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monomers and halide-based anions extracted the highest amount of mRNA compared to the

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other studied sorbent phases. The selectivity of the SPME approach for mRNA was enhanced by

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modifying the surface of a PA sorbent coating with oligo dT20 using carbodiimide-based amide

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linker chemistry,14 resulting in a dramatic improvement in mRNA extraction performance

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compared to an unmodified PA fiber. The mRNA extraction performance of the oligo dT20-

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modified PA sorbent coating was compared with a commercial spin column-based SPE

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extraction kit and demonstrated significant potential for the purification of mRNA from total

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RNA and yeast cell lysate samples. The results show that SPME-based RNA sample preparation

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provides a complementary approach to existing RNA purification methodologies and is

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particularly compatible with field sampling and point-of-care diagnostic applications.

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Experimental

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Real-time Reverse Transcription Quantitative PCR Assays. All materials and detailed

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experimental procedures for the preparation of total RNA and mRNA samples are provided in

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the Supporting Information. RNA analysis via RT-qPCR is a combination of two steps: (1)

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mRNA is first converted into complementary DNA (cDNA) utilizing RNA-dependent DNA

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polymerase at optimal isothermal conditions and (2) the obtained cDNA is then amplified using

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qPCR. The RT-qPCR assays employed for evaluating the mRNA extraction performance of

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SPME fibers were performed according to previously reported procedures, with minor


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modifications.15 Prior to generation of an external calibration curve, RT and qPCR steps were

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optimized separately. RT reactions were performed according to the following protocol: initial

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denaturation step of 5 min at 65 °C, followed by annealing of reverse primers (specific to beta-

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actin mRNA) at 4 °C for 10 min. The reaction temperature was then increased to 45 °C and held

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for 1 h to synthesize beta-actin cDNA utilizing SuperScript III reverse transcriptase. The cDNA

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was then analyzed by qPCR with a CFX96 Touch Real-Time PCR Detection System from Bio-

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Rad Laboratories (Hercules, CA, USA). The following thermal cycling protocol was employed

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for qPCR analysis: initial denaturation step of 5 min at 95.0 °C, followed by 40 cycles of 15 s at

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95.0 °C and 30 s at 67.0 °C. All amplification reactions were performed in triplicate. For each

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reaction, 1 µL of RT products were mixed with 19 µL of a reaction mixture consisting of 10 µL

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of SsoAdvanced Universal SYBR Green Supermix (2x), 0.6 µL of 10 µM forward and reverse

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primers, and 7.8 µL of deionized water. For the external calibration curve, an additional 75 mM

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and 15 mM NaCl was included in RT and qPCR steps, respectively. The quantification cycle

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(Cq) values obtained for amplification of beta-actin cDNA were used to indicate the amount of

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mRNA extracted by SPME fibers. All of the mRNA samples used in this study were treated with

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DNase I at 37 °C for 1 h to eliminate genomic DNA contamination and subsequently purified via

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isopropyl alcohol precipitation. Relevant control experiments to detect the genomic DNA and

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mRNA/cDNA contamination of RT-qPCR reagents were performed prior to mRNA

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quantification.

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A six-point external calibration curve (mass of mRNA varied from 1400 to 0.5 pg) was

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developed to quantify the amount of mRNA extracted by the SPME devices (Figure S1). The

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RT-qPCR amplification efficiency was calculated using Equation 1, where the slope is the slope

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of the linear regression within the calibration curve. The amplification efficiency of the beta-



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actin RT-qPCR assay was found to be 105.1%. Following RT-qPCR analysis, the amplification

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specificity and presence of primer dimers responsible for false positives were determined using

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high resolution melting point analysis. (

)

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Efficiency = [10 − 1] × 100

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Isolation and Purification of mRNA Using SPME Devices.

(1) Figure 1 shows the

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schematic workflow of SPME-based RNA analysis using RT-qPCR. Aqueous solutions (100 µL)

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of purified yeast mRNA (0.1 ng µL-1) were used to examine the applicability of SPME for RNA

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analysis. The mRNA was extracted by immersing the SPME fiber in the sample solution

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followed by a desorption step in NaCl to facilitate recovery of the nucleic acid from the sorbent

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coating. A 3 µL aliquot of the desorption solution was subjected to RT to generate cDNA from

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beta-actin mRNA using gene specific primers. An aliquot of RT products (4 µL) was used for

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qPCR amplification, resulting in a final NaCl concentration of 15 mM. Prior to subsequent

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extractions, the PIL sorbent coating was washed in 100 mM NaOH (100 µL) for 10 min. The

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chemical composition of the sorbent coating was regenerated by soaking in 2 M NaCl solution

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for 20 min.

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For the extraction of mRNA from total RNA, varying amounts (250 to 2000 ng) of total

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RNA were spiked into a 100 µL aqueous solution and PIL-based SPME was performed as

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described above. For extraction of mRNA from yeast cell lysate, approximately 3.0×107 S.

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cerevisiae cells (strain BY4735) were suspended in 400 µL of deionized water containing 0.25 g

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of acid washed glass beads. After vortexing the mixture for 10 min, 200 µL of the crude cell

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lysate suspension was added to 200 µL of deionized water and subjected to PIL-based SPME.

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Results and Discussion


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Investigating the Applicability of SPME for RNA Analysis. To understand the

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important structural features of sorbent coatings that can enhance the extraction of mRNA, a

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variety of SPME sorbent coatings were investigated including PILs (see structures and

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combinations in Figure 2A) and a commercially available PA sorbent phase. The fabrication of

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all PIL-based SPME devices was performed using procedures previously reported in the

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literature.16 Table S1 presents the thickness of all PIL sorbent coatings employed in mRNA

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extraction. The mRNA extraction performance of the different SPME sorbent coatings was

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evaluated by RT-qPCR of beta-actin mRNA from S. cerevisiae. Given that the quantity of cDNA

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determined by qPCR is proportional to the amount of mRNA extracted by the sorbent coating

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and subjected to RT, a lower Cq value from the qPCR assay indicates that a high amount of

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mRNA was extracted. When the qPCR amplification efficiency is 100%, a difference of one Cq

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value between two different samples indicates a two-fold difference in the amount of template

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cDNA.

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As shown in Figure 2B, the amount of mRNA extracted by the native PA fiber could not

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be calculated since the Cq values were higher than the calibration range, suggesting poor mRNA

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extraction capabilities of the sorbent coating. Recently, our group and others have exploited the

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unique extraction properties of PILs for DNA analysis.10-12 Although the chemical stability and

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molecular structure of RNA differs from DNA, it is reasonable to conceive that electrostatic

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interactions between the negatively charged phosphate back bone of RNA and the PIL cation

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framework may contribute to mRNA extraction. In order to identify important structural features

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of the PIL sorbent coating that can aid in RNA analysis, IL monomers substituted with different

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functional groups and crosslinkers were prepared as shown in Figure 2A. The effect of the PIL

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anion on RNA extraction performance was examined using fibers containing the same cationic



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composition with halide anions (Fiber 2) and bis[(trifluoromethyl)sulfonyl]imide [NTf2-] anions

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(Fiber 3). IL monomers and crosslinkers used in the fabrication of PIL-based SPME were

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synthesized and characterized using 1H NMR according to reported procedures from the

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literature.16,17

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Figure 2B shows the mRNA extraction performance of all PIL fibers and a commercial

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PA fiber employed in this study. The amount of mRNA extracted by the PIL sorbent coatings

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(Fiber 1 to 4) was significantly higher than the PA sorbent coating, suggesting that electrostatic

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interactions between the sorbent coating and RNA molecules are important for extraction.

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Among fibers with the same crosslinker composition (Fibers 1, 2, and 4), the incorporation of

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carboxylic acid groups within the IL monomer (Fiber 2) provided a higher extraction

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performance than Fiber 1 and 4 (with similar film thickness, as shown in Table S1). For

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example, the average Cq value of Fiber 1 was 31.07 ± 1.04, whereas Fiber 2 extracted a higher

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quantity of mRNA with Cq values of 28.92 ± 0.2. The replacement of halide anions (Fiber 2)

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with [NTf2-] anions (Fiber 3) resulted in a 2.18 cycle increase in the Cq value, indicating a

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dramatic decrease in mRNA extraction performance, suggesting the crucial role that halide

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anions play in the extraction.

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In addition to electrostatic interactions, the ion-exchange mechanism between anions of

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the PIL sorbent coating and the negatively charged phosphate backbone of mRNA may also

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contribute to extraction (as previously observed for DNA extraction).10,12 To test this hypothesis,

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a non-exchangeable styrenesulfonate anion component was incorporated into the PIL sorbent

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coating (Fiber 5). Previous studies demonstrated that co-polymerization of p-styrenesulfonate

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anions with PIL cation moieties provided a stable sorbent phase that was incapable of

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undergoing anion exchange in high ionic strength solutions (30% w/v NaCl) and these results


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were validated by measuring the sulfur composition of the sorbent phase via elemental

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analysis.18 Figure 2 shows that the amount of mRNA extracted by Fiber 5 was significantly

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lower than all other PIL-based sorbent coatings (Fiber 1-4), indicating that an ion-exchange

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mechanism plays a significant role in mRNA extraction by PIL sorbent coatings.

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Optimization of Extraction and Desorption Parameters for PIL-based SPME.

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Because of its high extraction performance and good reproducibility (0.7 % RSD for Cq values)

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compared to other PIL fibers, extraction conditions were optimized using Fiber 2. Initially, the

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effect of NaCl concentration on mRNA recovery was evaluated. As shown in Figure S2, the

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amount of mRNA recovered from the PIL sorbent coating at a constant desorption time of 30

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min increased as the NaCl concentration was increased from 0 to 500 mM. However, beyond

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500 mM, no significant enhancement in mRNA recovery was observed. The desorption volume

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was selected such that the lowest volume of solution was used that would fully immerse the PIL

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sorbent coating (i.e., 50 µL). A 3 µL aliquot of desorption solution (500 mM NaCl) was used for

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RT-qPCR analysis, corresponding to 75 mM and 15 mM of NaCl in RT and qPCR steps,

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respectively. At higher volumes of desorption solution (>75 mM NaCl in RT), the

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reproducibility of the RT-qPCR assay was severely compromised (data not shown).

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In an effort to minimize carry-over for the SPME method, a 10 min washing step using

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10 mM NaOH (pH 10-11) was employed to remove residual RNA from the PIL sorbent coating

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(Fiber 2) by well-established base-catalyzed RNA hydrolysis.19 Since PILs can undergo ion-

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exchange interactions in high ionic strength solutions,18 the sorbent coating was subsequently

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regenerated by exposure to a 2 M NaCl solution for 20 min. Figure S3 shows representative RT-

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qPCR plots for wash fractions prior to the subsequent extraction as well as the desorption

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fraction following extraction of mRNA using the regenerated sorbent coating (Fiber 2). These



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results demonstrate the chemical resistance as well as the reusability of PIL-based SPME devices

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compared to expensive, single-use commercial silica-based SPE kits for RNA analysis.

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The sorption-time profile of Fiber 2 was evaluated by varying the extraction time from 5

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to 60 min under agitation at 80 rpm using an orbital shaker. From Figure S4, the amount of

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mRNA extracted by the PIL sorbent coating increased dramatically from 5 to 45 min, but no

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obvious improvement in extraction was observed from 45 to 60 min. Although the highest

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amount of mRNA was extracted at 45 min, an extraction time of 30 min was chosen for all

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subsequent experiments in order to minimize the overall analysis time. Under optimized

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extraction conditions, the fiber-to-fiber reproducibility for Fiber 2 was evaluated (using three

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fibers of Fiber 2) and resulted in Cq values with a RSD of 3.2%.

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Extraction of mRNA from Total RNA Samples and Yeast Cell Lysate. Total RNA is

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comprised of a pool of various RNA molecules in a cell (e.g., mRNA, ribosomal RNA, transfer

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RNA). In a typical eukaryotic cell, the quantity of mRNA ranges from 1-2% of the total RNA.20

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As shown in Figure S5, the amount of mRNA extracted by Fiber 2 was extremely small at 2.5

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and 5.0 ng/µL of total RNA and the obtained Cq values were out of the calibration range. This

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could be either due to the low amount of mRNA available for extraction or high interference

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from background total RNA. By increasing the amount of total RNA from 5 to 10 ng/µL, a

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drastic improvement in extraction performance was observed. However, no significant

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enhancement in extraction was observed beyond 15 ng/µL of total RNA, suggesting a strong

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effect of background RNA on mRNA extraction. It is important to note that the total RNA

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concentration range where PIL-based SPME can be operated is at least one order of magnitude

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lower than what is typically used for direct RT-qPCR analysis. Encouraged by these results, the

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applicability of PIL-based SPME was also investigated for its ability to isolate beta-actin mRNA


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from the crude lysate of approximately 3.0×107 S. cerevisiae cells. The PIL-based SPME method

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was also compared with a conventional phenol/chloroform LLE method. As shown in Figure S6,

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the amount of mRNA extracted using PIL-based SPME (Cq: 27.56 ± 0.75) was significantly

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higher than the LLE method (Cq: 32.72 ± 1.51), with superior reproducibility (indicated by the

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lower standard deviation in Cq values) and minimal sample processing (centrifuge and organic

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solvent-free).

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Selective

mRNA

Extraction

Using

Oligo

dT20-Modified

PA

Fiber.

The

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functionalization of extraction media with oligo dT20 (poly T) capable of specifically hybridizing

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with the poly A tail of mRNA molecules represents the most common strategy employed in

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selective mRNA analysis.5,21 The development of PIL sorbent coatings for selective mRNA

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analysis in the presence of total RNA would be challenging due to the nonspecific interactions

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between total RNA and the PIL sorbent coating (on basis of data from Figure S5) that may result

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in difficulty quantifying low abundance mRNA samples. In an attempt to reduce the interference

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from background total RNA as well as to improve the selectivity of the sorbent coating toward

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mRNA, the PA sorbent coating was selected for oligo dT20 modification. Since the PA sorbent

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phase lacks ion-exchange and electrostatic interactions with mRNA molecules (on basis of data

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from Figure 2B), these types of interactions between the PA fiber and total RNA may also be

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minimal and result in reduced interference for mRNA extraction. Importantly, the presence of

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carboxylate functional groups within the acrylate polymer matrix makes it a suitable substrate for

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oligo dT20 modification via carbodiimide-based amide linker chemistry.

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Figure 3 shows the comparison of RT-qPCR amplification plots following SPME of

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aqueous mRNA samples using oligo dT20-modified and unmodified PA fibers (detailed

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procedures in Supporting Information). The high mRNA extraction performance exhibited by the



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modified PA fiber (Cq: 33.74 ± 0.24; n=3) compared to the unmodified PA fiber (Cq: 39.14)

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could be due to hybridization between the poly A tail of mRNA and oligo dT20. Under identical

267

conditions (5 ng µL-1 total RNA), the PIL-based sorbent did not extract sufficient mRNA from

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the total RNA sample for RT-qPCR analysis. It is worthwhile to mention that the concentration

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range investigated to demonstrate the selectivity of the SPME approach for mRNA was

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approximately four-fold lower than what was reported in previous studies.15 The selectivity of

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the modified PA sorbent coating toward mRNA was also examined in the presence of total RNA

272

(5.0 ng/µL in 100 µL deionized water) and its extraction performance compared against a

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commercial SPE kit. RT-qPCR analysis (Figure S7) revealed that the average Cq value for

274

modified PA sorbent coating was 32.67 ± 1.31 (n=3), whereas the commercial SPE kit provided

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a Cq value of 28.93 (triplicate experiments were not performed since the extraction media is not

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reusable according to manufacturer’s guidelines). The low Cq value of the commercial SPE kit

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indicates that a higher mass of mRNA was extracted when compared to modified PA sorbent

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coating. Nevertheless, the modified PA-based SPME method preconcentrated a sufficient

279

quantity and quality of mRNA for the RT-qPCR assay with low analysis time and minimal

280

experimental setup (i.e., without a centrifugation apparatus, binding or elution buffers essential

281

for SPE kits, as shown in Table S2). Furthermore, the oligo dT20 modified PA-based SPME

282

method was applied for the extraction of mRNA from crude cell lysate of approximately 3.5×107

283

S. cerevisiae cells. The developed method yielded a Cq value of 26.12 ± 1.21 (n=3),

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demonstrating its capability to extract sufficient quantity and quality of mRNA from complex

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sample matrices. High-resolution melting point analysis of RT-qPCR products confirmed the

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amplification specificity and purity of the extracted mRNA, as shown in Figure S8.


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The fiber-to-fiber reproducibility of 5'-/5AmMC12/dT20-3' loading on PA fibers was

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examined using a microplate fluorescence assay (Figure S9, assay procedure described in

289

Supporting Information). Based on the fluorescence assay, approximately 40.39 and 25.34 ng of

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oligo dT20 was immobilized on two different PA fibers, indicating a significant difference in

291

their surface composition. This could be due to lot-to-lot variation in the production of PA fibers,

292

which can result in a difference in the amount of surface acid groups available for the aminolysis

293

reaction. Ongoing studies in our lab are focused on developing new carboxylic acid-based SPME

294

sorbent coatings within a non-ionic polymer matrix in an effort to minimize nonspecific

295

interactions with background total RNA.

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In summary, the feasibility of SPME-based RNA purification was investigated using a

297

variety of sorbent coatings. The optimized PIL-based SPME method demonstrated superior

298

performance in extracting mRNA from crude yeast cell lysate samples compared to

299

phenol/chloroform LLE. The reusability and chemical stability of PIL sorbent coatings constitute

300

important advantages over expensive, single-use commercial silica-based SPE kits. The

301

selectivity of SPME toward mRNA was enhanced by exploiting an oligo dT:mRNA

302

hybridization approach. The modified PA-based SPME method was capable of isolating mRNA

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from low quantities of total RNA samples with low analysis time and minimal experimental

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setup compared to commercial SPE kits.

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Acknowledgements

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The authors acknowledge funding from Chemical Measurement and Imaging Program at the National Science Foundation (Grant number CHE-1413199).

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Supporting Information

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Figures S1-S10, materials and measurements, preparation of RNA samples, oligo dT20

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functionalization of PA fibers, microplate fluorescence assay, Table S1 and S2, mRNA

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preconcentration using PA fibers. This information is available free of charge via the Internet at

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http://pubs.acs.org/.

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References

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1. Alwine, J. C.; Kemp, D. J.; Stark, G. R., Proc. Natl. Acad. Sci. USA 1977, 74, 5350-5354. 2. Gilman, M., Current protocols in molecular biology 1993, 4.7. 1-4.7. 8. 3. Schmittgen, T. D.; Livak, K. J., Nat. Protoc.2008, 3, 1101-1108. 4. Fleige, S.; Pfaffl, M. W., Mol. Aspects. Med. 2006, 27, 126-139. 5. Wen, J.; Legendre, L. A.; Bienvenue, J. M.; Landers, J. P., Anal. Chem. 2008, 80, 6472 6479. 6. Arthur, C. L.; Pawliszyn, J., Anal. Chem. 1990, 62, 2145-2148. 7. Wang, Y.; Schneider, B. B.; Covey, T. R.; Pawliszyn, J., Anal. Chem. 2005, 77, 8095-8101. 8. Goryński, K.; Kiedrowicz, A.; Bojko, B., J. Pharm. Biomed. Anal. 2016, 127, 147-155. 9. Vuckovic, D.; de Lannoy, I.; Gien, B.; Shirey, R. E.; Sidisky, L. M.; Dutta, S.; Pawliszyn, J., Angew. Chem., Int. Ed.,2011, 50, 5344-5348. 10. Wang, X.; Xing, L.; Shu, Y.; Chen, X.; Wang, J., Anal. Chim. Acta 2014, 837, 64-69. 11. Nacham, O.; Clark, K. D.; Anderson, J. L., Anal. Methods 2015, 7, 7202-7207. 12. Nacham, O.; Clark, K. D.; Anderson, J. L., Anal. Chem. 2016, 88, 7813-7820. 13. Rogacs, A.; Qu, Y.; Santiago, J. G., Anal. Chem. 2012, 84, 5858-5863. 14. Wang, C.; Yan, Q.; Liu, H.-B.; Zhou, X.-H.; Xiao, S.-J., Langmuir 2011, 27, 12058-12068. 15. Nestorova, G. G.; Hasenstein, K.; Nguyen, N.; DeCoster, M. A.; Crews, N. D. Lab Chip 2017, 17, 1128-1136. 16. Ho, T. D.; Toledo, B. R.; Hantao, L. W.; Anderson, J. L., Anal. Chim. Acta 2014, 843, 18-26. 17. Baltazar, Q. Q.; Chandawalla, J.; Sawyer, K.; Anderson, J. L., Colloid Surf. A 2007, 302, 150-156. 18. Feng, J.; Sun, M.; Xu, L.; Wang, S.; Liu, X.; Jiang, S., J. Chromatogr. A 2012, 1268, 16-21. 19. Oivanen, M.; Kuusela, S.; Lönnberg, H., Chem. Rev. 1998, 98, 961-990. 20. Iyer, V.; Struhl, K. Proc. Natl. Acad. Sci. USA, 1996, 93, 5208-5212. 21. Lee, H.; Jung, J.; Han, S.; Han, K., Lab Chip 2010, 10, 2764-2770.


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Figure 1. Schematic illustration of RNA analysis using the SPME approach coupled with reverse transcription quantitative polymerase chain reaction (RT-qPCR). 345

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Fiber

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1

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Monomer

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Cross-linker

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Mass of extracted mRNA (pg)

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B Quantification cycle

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∗

∗

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Figure 2. (A) Chemical composition of PIL-based SPME sorbent coatings examined in this study; (B) Effects of sorbent coating composition on the mass of mRNA extracted. Concentration of mRNA: 0.1 ng µL-1; sample solution: deionized water (100 µL); desorption time: 30 min; extraction time: 10 min; desorption solvent: 1 M NaCl; desorption solvent volume: 50 µL; ( ) represents quantification cycles; ( ) denotes mass of extracted mRNA. Extractions were performed in ∗ triplicate (n=3). Mass of mRNA extracted by PIL Fibers 5 and a commercial polyacrylate fiber was not determined as the Cq values obtained were out of the calibration range. 375 376 377 378 379 380


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Figure 3. Representative RT-qPCR amplification plot following extraction of mRNA using (A) oligo dT20-modified PA fiber and (B) unmodified PA fiber. Extractions were performed in triplicate (n=3); concentration of mRNA: 0.1 ng µL-1; sample solution: 100 µL deionized water containing 100 mM NaCl; desorption solvent: 30 µL deionized water. ( ) oligo dT20 complementary to poly A tail of mRNA; ( ) mRNA with poly A tail. 394 395

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Rapid and Selective RNA Analysis by Solid-Phase Microextraction

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