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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

264

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



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Page 12 of 18

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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

268

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

273

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

275

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

276

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

277

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

278

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),

284

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

285

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

288

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

290

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

303

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

304

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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Selective and Efficient RNA Analysis by Solid ... - ACS Publications

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