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Absolute quantification of RNA molecules using fluorescence correlation spectroscopy with certified reference materials Akira Sasaki, Johtaro Yamamoto, Masataka Kinjo, and Naohiro Noda Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02213 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 21, 2018
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Analytical Chemistry
Absolute quantification of RNA molecules using fluorescence correlation spectroscopy with certified reference materials Akira Sasaki1, Johtaro Yamamoto1, 2, Masataka Kinjo2 and Naohiro Noda1* 1Biomedical
Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan, 2Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido 001-0021, Japan. ABSTRACT: The accuracy and precision of quantification values of biomolecules, such as nucleic acids, are critical for the reliability of biomedical research and clinical examinations. To obtain an accurate quantitative value, it is necessary to use a measurement standard that has the same sequence and length as the target gene. The absence of an appropriate measurement standard leads to uncertain results. The development of a wide variety of different kinds of measurement standards, which have different sequences and lengths, is time-consuming and troublesome. We employed fluorescence correlation spectroscopy (FCS), which can be used to count the molecular number (absolute concentration) regardless of the molecular size and shape, without a standard curve. The confocal volume (i.e., the volume of excitation laser focus) of the FCS system was calibrated by measuring the primary standard of the fluorescent material. Furthermore, we investigated how to avoid artifacts originating from systematic aberrations or sample conditions. We validated the RNA concentration obtained from our FCS measurements using another primary standard RNA solution as a sample. Here, we describe an FCS calibration procedure with fluorescein solution standard reference material (SRM) 1932 as a primary standard and cross-validation of FCS values using RNA solutions certified reference material (CRM) 6204-a. The established method was applied to determine the concentrations of RNA samples that can be used as a laboratory working standards. The FCS method with a characterized SRM and CRM should serve as a universal method for absolute quantification of the number of biomolecules.
Accurate and precise quantification of biomolecules, such as nucleic acids and proteins, is important for clinical examinations, biomedical research, and environmental assessments. Quantification of nucleic acids has been conventionally performed by ultraviolet (UV) adsorption1, fluorescence spectrometry2-3, and real-time polymerase chain reaction (PCR)4. These methods determine a quantitative value by assuming absorptivity coefficients or preparing a standard curve after measuring samples with known concentrations. For this reason, well-characterized measurement standards of nucleic acids are needed. A wide variety of different kinds of gene targets with different sequences and lengths must be quantified for each diagnostic test. A limitation of current methods is that the quantified values are affected by the nucleic acid length and sequence, so the use of unmatched measurement standard leads to uncertain results. A molecular numberbased absolute-measurement technique would be ideal to resolve this issue. Therefore, a method for counting the absolute number of molecules independently of the molecular characteristics (such as the nucleic acid length and sequence) is desired. We employed fluorescence correlation spectroscopy (FCS)5-6 for absolute quantification of nucleic acid samples. FCS is based on the analysis of fluorescence-intensity fluctuations of laser-excited fluorescent molecules in
a confocal observation volume and has been widely applied for measuring molecular dynamics and molecular interaction, and for quantifying molecular concentrations in solution and in living cells7-9. The key feature of FCS is that the method can count the number of molecules (absolute concentration) regardless of the molecular size and shape. From FCS measurements, the number of molecules in the confocal volume is obtained. Therefore, determination of the size of the confocal volume is required to convert the number of molecules into the concentration. However, accurate determination of the effective confocal volume is challenging because of the intensity distribution of the illumination profile. Currently, several methods have been proposed to estimate the confocal volume6, 10-11. A commonly used approach is to estimate the effective confocal volume by diffusion analysis of a molecule with a known diffusion coefficient, such as rhodamine 6G12 assuming that the volume shows a Gaussian profile; however, this can be problematic in cases where the focused laser profile is not Gaussian, in particular, when using a high-numerical aperture (NA) objective lens10. The error in the confocal volume estimation that originates from collapse of the Gaussian approximation causes significant errors in determining molecular concentrations by FCS.
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In this work, we determined the confocal volume without using the Gaussian approximation by measuring fluorescein solution National Institute of Standards and Technology (NIST) standard reference material (SRM) 1932, the concentration of which is determined with a certified value. The approach allowed us to achieve highly accurate calibration of the effective FCS confocal volume. Further, the optical-saturation effect13-15 is another factor that hinders accurate FCS measurements. The opticalsaturation effect becomes an issue when the excitation light intensity becomes so large that the excitation intensity increase does not lead to a proportional increase in the emission fluorescence intensity. When performing measurements, it is difficult to know how optical saturation affects a measurement due to the lack of a true quantitative value. Furthermore, FCS is generally sensitive to artifacts such as non-ideal, laser-illumination conditions and refractive index mismatches16. Therefore, the reference measurement of standards, which have certified concentrations, are important for discerning potential artifacts of FCS measurements and eliminating them with confidence. Here, we describe FCS calibration with fluorescein solution (NIST SRM 1932) and cross-validation of FCS values using RNA solutions (National Metrology Institute of Japan (NMIJ) Certified Reference Material (CRM) 6204a. Further, application of the established absolute-quantification method for determining the concentration of an RNA sample, which can be used as a laboratory-working standard, is reported. We also investigated how to avoid artifacts originating from systematic aberrations or sample conditions. MATERIALS AND METHODS Materials Fluorescein solution NIST SRM193217 was purchased from NIST (Gaithersburg, MD, USA). RNA solutions NMIJ CRM6204-a18 was obtained from NMIJ, National Institute of Advanced Industrial Science and Technology (Ibaraki, Japan). The Quant-iT RiboGreen RNA Assay Kit and PowerSYBR Green RNA-to-CT 1-Step Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Preparation of RNA solutions RNA CRM6204-a was diluted 10 times gravimetrically in TE buffer (pH = 7.4). Fluorescence staining of RNA was performed by mixing diluted RNA solutions with RiboGreen working solutions (×500 in TE buffer) at a 1:1 ratio, followed by a 5-min incubation at room temperature in the dark. A synthetic 906-base pair (bp) DNA fragment (see the sequence in the Supporting Information section) containing the sequence of Homo sapiens Wilms tumor 1 (WT1), transcript variant B (GenBank accession No.
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BC032861)19, and the T7 promoter, was designed and produced by GeneArt Strings DNA Fragments, Thermo Fischer Scientific (Waltham, MA, USA). WT1 RNA was transcribed from the DNA fragment in vitro using the MEGAscript T7 Kit (Life Technologies, Carlsbad, CA, USA) and purified using the RNeasy MinElute Cleanup Kit (Qiagen, Düsseldorf, Germany). Electrophoresis of the RNA was performed using an Agilent 2200 TapeStation system and an RNA ScreenTape (Agilent, SantaClara, CA, USA). The WT1 RNA was also stained with RiboGreen for FCS measurements. FCS experiments FCS analyses were performed on the FCS Compact BL system (Wako Pure Chemical Industries, Ltd., Osaka, Japan) with a modified setup to reduce the excitation laser power (473 nm). The laser powers were estimated from laser output (1 mW) and transmission rate of neutral density (ND) filters. The laser power was measured by using a Microscope Power Sensor (S170C; Thorlabs, Inc., NJ, USA) and an Optical Power and Energy Meter Console (PM400; Thorlabs, Inc.). The actual laser output was confirmed as 1.08 mW at the sample position. When using ND filters with a 0.1% transmission rate, the laser power was 1.05 µW, as expected. The system was equipped with a water-immersion objective lens (UApoN 340, 40×, NA = 1.15; Olympus Corporation, Tokyo, Japan). Measurements were taken at room temperature (25°C) in microwell slides (Wako Pure Chemical Industries Ltd., Osaka, Japan) or 8-well Nunc chambered coverglasses (Thermo Fisher Scientific, Waltham, MA, USA). The glass surfaces of the wells were treated with 20% N101 blocking reagent (NOF corporation, Tokyo, Japan). Each sample solution (20 µl for microwell slides, 50 µl for Nunc coverglasses) was placed on the glass surface of a well and measured for 30 s at 200 µm above the glass surface. The data analysis was performed using Hamamatsu Photonics Control FCS software. The fluorescence autocorrelation function G (t) was calculated using the following equation: 𝐺(𝜏) =
〈'(()∙'((*+)〉 〈'(()〉-
(1)
where t denotes the time delay, I is the fluorescence intensity detected in the laser-illuminated observation volume (confocal volume), and G (t) denotes the autocorrelation function. Acquired autocorrelation curves were fitted to a onecomponent model as follows:
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Analytical Chemistry
G(τ) = 1 +
234*4567(3+⁄+8 ) :(234)
;1 +
+ +
- +
