key: cord-0929488-hh28rfic
authors: Niu, Chunyan; Dong, Lianhua; Yunhua, Gao; Zhang, Yongzhuo; Wang, Xia; Wang, Jing
title: Quantitative Analysis of RNA by HPLC and Evaluation of RT-dPCR for Coronavirus RNA Quantification
date: 2021-02-22
journal: Talanta
DOI: 10.1016/j.talanta.2021.122227
sha: fd297028375a0091cfca9c6cfa35fdcc5bd06105
doc_id: 929488
cord_uid: hh28rfic

Nucleic acid detection and quantification have been known to be important at various fields, from genetically modified organisms and gene expression to virus detection. For DNA molecules, digital PCR has been developed as an absolute quantification method which is not dependent on external calibrators. While when it comes to RNA molecules, reverse transcription (RT) step must be taken before PCR amplification to obtain cDNA. With different kinds of reverse transcriptase (RTase) and RT reaction conditions being used in laboratory assays, the efficiency of RT process differs a lot which led variety in quantification results of RNA molecules. In this study, we developed HPLC method combined with enzymatic digestion of RNA to nucleotides for quantification of RNA without RT process. This method was metrologically traceable to four nuceloside monophosphate (NMP) Certification Reference Materials of National Institute of Metrology, China (NIMC) for insurance of accuracy. The established method was used to evaluate the reverse transcription digital polymerase chain reaction (RT-dPCR) of three target genes of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) RNA, including ORF1ab, E and N gene. Three available RT kits had been evaluated and disparities were observed for the RT efficiency varied from 9% to 182%. It is thus demonstrated that HPLC combined with enzymatic digestion could be a useful method to quantify RNA molecules and evaluate RT efficiency. It is suggested that RT process should be optimized and identified in RNA quantification assays.

Nucleic acid detection and quantification play a crucial role at various fields, such as genetically modified organisms [1] , gene expression and regulation [2] [3] [4] [5] and virus detection [6] [7] [8] [9] [10] . Ribonucleic acid (RNA) is the genetic material of RNA virus, which may cause many human diseases, including the most recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has spread rapidly all over the world [11, 12] . The pathogen was identified as a novel coronavirus, which belongs to the family Coronaviridae [12, 13] . There has been two major epidemics caused by members of the coronaviruses in history, including severe acute respiratory syndrome coronavirus (SARS-CoV) occurred since 2003 [14, 15] , and Middle East respiratory syndrome coronavirus (MERS-CoV) occurred since 2012 [16] . For RNA measurement, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) has been considered as the primary method and found wide applications. Reverse transcription digital polymerase chain reaction (RT-dPCR), due to its sensitivity and accuracy, has also been developed and used in RNA quantification [8, 17] . RT-dPCR allows absolute quantification of cDNA without calibrant by partitioning the PCR solution into small reactions and applying Poisson statistics to the proportion of the negative partitions [18, 19] . However, all PCR-based detection methods of RNA are dependent on reverse transcription (RT) of RNA to cDNA, which is essential prior to J o u r n a l P r e -p r o o f PCR but may induce biases to the results [20] . This fact has been noticed but often neglected, for example, in the expression levels of mRNA and its clinical significance where variability up to100-fold were observed [20] [21] [22] [23] . The variability depends on the choice of reverse transcriptase (RTase) as well as the reaction conditions like temperature and priming strategy (oligo (dT), random hexamer, or gene specific primer) [21] [22] [23] [24] [25] .

Thus, in this study, we focus on developing accurate RNA quantification method not depending on reverse transcription process. Most regularly used method like ultra-violet (UV) and fluorescence spectroscopy can be simple but lack of specificity and traceability to quantify RNA, due to the interference of other impurities such as organics, proteins or DNA [26, 27] . Snake venom phosphodiesterase I (SVP) has been used in quantification of 20mer oligonucleotide by digesting DNA to deoxynucleotide monophosphates (dNMPs) followed by isotope dilution mass spectrometry (IDMS) [28, 29] , and ultrasonic treatment was needed prior to SVP digestion for quantification of large dsDNA [30] . RNA can be hydrolyzed to nucleotide monophosphates (NMPs) by SVP whereas RNA quantification method of this kind has not been reported to date as we know.

The purpose of this study is to investigate the ability of digestion of RNA by SVP followed by high performance liquid chromatography (HPLC) to quantify RNA molecules using four NMP Certification Reference Materials obtained from National Institute of Metrology, China (NIMC) as calibrators for insurance of accuracy. The established method was applied to quantify an in vitro transcribed MERS-RNA

An in vitro transcribed RNA molecule consisting of three genes of MERS (partial ORF1ab, full length E and N) was used in the whole study. Gene synthesis and construct containing MERS-1ab-E-N were ordered from Sangon Biotech Co., Ltd (Shanghai, China). Plasmid containing synthetic genes was linearized with BamH at the 3'-end and purified as the linear template DNA. In vitro transcription reaction was performed using MEGAscript™ T7 Transcription Kit (Thermo Fisher Scientific, USA) according to the manufacturer's suggested protocol, and 1 μL of TURBO DNase were added after transcription to remove the template DNA. Transcribed RNA was purified with MEGAclear™ Kit (Thermo Fisher Scientific, USA). Purity of the RNA transcript was measured using the RNA 6000 Pico kit and analyzed on a 2100

Bioanalyzer Instrument using their 2100 Expert Software (Agilent Technologies, USA). RNA stock solution was diluted to 1.2 ng/μL with RNA storage solution (Thermo Fisher Scientific, USA), aliquoted to 100 μL/tube, stored at -80 , and used as RNA template throughout this study.

Four Certified Reference Materials of adenosine 5'-monophosphate (AMP,

A total volume of 100 μL mixture containing 90 μL of RNA, 9 μL of SVP buffer (100Mm Tris-HCl pH 8.8, 10mM ammonium acetate, and 100 mM Magnesium acetate), and 1 μL of SVP (0.00023 U/μL) were incubated at 37 for 15 min. No enzyme control (NEC) and no template control (NTC) were prepared by adding water in place of enzyme or template RNA.

The HPLC system of Agilent 1200 series was used in this work. The separation of the four nucleotides was achieved using an SB-AQ C 18 J o u r n a l P r e -p r o o f

The primer and probe sequences for detecting ORF1ab and E gene target of the MERS-CoV published by world health organization (WHO) were used for this study [31] . For detecting N gene target, primer and probe published by Center for disease control and prevention (CDC) was used [32] .

One were the same as above except that the RT process was omitted. The primers and probes and the reaction conditions were identified to be suitable when cDNA was used in dPCR assays.

SB-AQ is a reversed-phase packing which had been used for separation of dNMP [30] UMP and GMP, and data at 280nm was chosen for CMP, because the signal intensity of CMP was remarkably higher at 280nm than at 260nm.

The total sequence length of the in vitro transcribed RNA, including the transcription initiation site of T7 RNA polymerase, was 4610 nucleotides according to the synthetic report of manufacturer. Purification of transcribed RNA was applied to remove nucleotides, proteins and salts. Purity and homogeneity of the transcribed RNA were checked and confirmed through chip assay where only one band exited.

When it came to DNA, long fragment nucleic acid could not be completely digested by using only enzymes [30] . However, as single-stranded RNA is sensitive to RNase and degrades more easily, it was deduced that the in vitro transcribed RNA could be digested completely by using SVP without any pretreatment. In this study, digestion optimization was carried out with the peak area of each NMP analyzed at time point of 5min, 15min, 30min, 60min and 120min. Over the 2 h time course, no significant change was observed in the amount of hydrolysis products from 15 min to 1 h, yet slight reduction appeared at 2 h ( Fig S1) . Furthermore, in the chromatography result of HPLC, the profile of digestion products released from RNA sample was comparable with the that of NMP calibrators ( Fig 1B) . Therefore, RNA was considered to be completely digested with hydrolysis time of 15 min to 1 h at 37 .

Further hydrolysis of nucleotides may occur after 2 h.

In view of the dependence of the accuracy of our analysis method on calibrators, they were gravimetrically prepared and metrologically traceable to CRMs of NIM 

Partial ORF1ab, full length E and N gene of MERS were constructed onto the same plasmid to evaluate the amplify and reverse transcription efficiency of the RT-dPCR assays. Primers and probes sequence used in this study origin from former publications [31, 32] . Optimization for dPCR has been carried out including primers and probes concentration and annealing temperatures on Bio-rad QX200 platform using one-step RT-dPCR kit. Validation experiments were performed to assess the performance characteristics of the RT-dPCR including repeatability, linearity, specificity and sensitivity of the assay (supplemental Fig S3 and Table S1 .)

The in vitro transcribed RNA was 10-fold diluted into RNA storage buffer (Thermofisher) and measured. Good linearity was observed across the dilution range from approximately 10 5 to 10 0 copies/reaction for the three target genes with R 2 ≥ 0.9999. The LoQ of the RT-dPCR assay was assess to be 64 copies/reaction for ORF1ab ,167 copies/reaction for E and 156 copies/reaction for N gene, with a criterion of CV value lowering than 25%.

The specificity of the assays for three genes has been tested in the previous report [31, 32] . In this study, we further validated that the assays had no positive results when testing the SARS-CoV-2 RNA reference material which was prepared by National institute of Metrology, China.

The transcribed MERS-RNA solution prepared in this study was quantification by RT-dPCR using the established assays. Additional to one-step RT-dPCR kit of Bio-rad, two cDNA synthesis kits of Thermofisher mentioned above were used to generate cDNA, and supermix for probes of Bio-Rad was subsequently used to J o u r n a l P r e -p r o o f quantify the cDNA. For one-step RT-dPCR, three tubes of RNA were tested with triplicate assays for each tube; for two-steps RT-dPCR, three tubes of RNA were tested with each tube being reverse transcribed respectively and triplicate assays of dPCR were performed for each cDNA. The quantification results were showed at Table 2 , we noticed dramatically disparities in the quantification results and RT efficiency among different methods. The efficiency of One-step RT-dPCR kit and Superscript kit was range from 9% to 71%, and that of Superscript was more than 100%. The reverse transcriptase in both the two ThermoFisher kits are MMLV RT and different reaction conditions have been optimized and developed by manufacture for them. For Superscript kit, the reverse primers existed in the mix of RT reaction including both oligo(dT) 20 and random hexamers and other components. Its suggested reaction temperature was 50 , and higher temperatures up to 60 was suggested for difficult templates. But we didn't get higher yields of cDNA at 60 than at 50 . For Superscript kit, a mix of template RNA, dNTP and the reverse primers was heated at 65 for 5min followed by incubation on ice for at least 1min to get the annealed RNA prior to RT reaction.

We suspected non-specific amplification occurred during this reaction leading to excessive quantification result.

For one-step RT-dPCR, the reverse transcriptase was integrated within one tube with PCR reactions. Its efficiency was higher than Superscript kit for both E and N gene, but lower for ORF1ab gene. Because the three genes of MERS-RNA were constructed to one single strand, their copy number concentrations were theoretically expected to be the same. Nevertheless, different quantification results and RT efficiency were observed for the three genes, no matter which kit was used. This may due to the variability of G-C content or secondary structures of the RNA template.

As indicated above, absolute quantification of RNA via RT-dPCR relies heavily upon the efficiency of the reverse transcription process. For accurate quantification of RNA molecules, work flow of optimization should be to designed to identify the most appropriate RT type and reaction conditions for target genes.

J o u r n a l P r e -p r o o f

A quantification method of RNA molecules with no need of reverse transcription by using enzymatic digestion followed by HPLC was successfully applied for the quantification of in vitro transcribed MERS-RNA. This method was demonstrated to be accurate for the metrological traceability to certification reference materials.

RT-dPCR method of MERS-RNA were established and evaluated through the HPLC result and dramatically disparities of RT efficiency were observed, thus RT type and reaction conditions were recommended to be optimized and identified.

The 

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