RNA Extraction from Viral Samples

The extraction of RNA from viral samples is an important step for pathogen research. Here we present a demonstrated RNA extraction method from viral samples.

Purpose

Viral nucleic acid extraction is the first step for downstream genomic analysis by RT-PCR. Here we present a research study at Miami Cancer Institute in collaboration with Beckman Coulter Life Sciences using the RNAdvance Viral research reagents described in the Viral Nucleic Acid extraction from swabs using the RNAdvance Viral extraction kit instructions for use. We demonstrate use of the viral RNA extracted with the RNAdvance Viral reagents within our RT-PCR workflow. The data presented includes the reagent's analytical performance and concordance data against a competitor's extraction kit by comparing qRT-PCR Ct values from universal transport media input material.

Workflow Overview

RNAdvance Viral reagents is an RNA isolation chemistry that uses Solid Phase Reversible Immobilization (SPRI) technology. It enables the generation of highly purified RNA with demonstrated compatibility with up to 200 μL of sample input. The extraction for 24 samples processed manually takes about 1 hour. For high throughput processing, two plates (192 samples) can be run in 1.75 hours using a Biomek i5 Extraction Solution with minimal human interactions. The RNAdvance Viral workflow (Figure 1) consists of sample lysis and bind-wash-elute steps.

RNA Isolation from Viral Samples Workflow

Data presented was produced independently by the Miami Cancer Institute.

1. Estimation of Analytical Performance - Sensitivity

Exact Diagnostics SARS-CoV-2 Standard positive (RUO, EDX, cat# COV019) and negative (EDX, cat# COV000) controls were spiked into the pooled negative universal transport media (HEALTHLINK, cat# 330C.DHI) at 200, 20, 2, and 0.2 copies/μL. RNA was extracted manually using either Beckman Coulter RNAdvance Viral reagents or another bead-based extraction kit (Competitor A). qRT-PCR assays were carried out using Integrated DNA Technologies (IDT) 2019-nCoV RUO kit.

Ct values were assessed via qRT-PCR using research primer sets targeting the SARS-CoV-2 nucleocapsid N1, N2, and N3 fragments (N1, N2, and N3) for the detection of viral RNA and the RNase P (RP) primer set for the detection of human RNase P RNA. Positive controls 2019-nCoV_N_Positive Control (IDT: 10006625) and qPCR negative controls (NTC Ambion cat# AM4932) were included to ensure proper testing control. Exact Diagnostics RNA Standard (SARS-CoV-2 Standard) with no extraction was used for recovery efficiency control.

Data showed in the Ct value is comparable between the RNAdvance Viral reagents and the Competitor A extraction kit (Table 1). RNA that extracted using RNAdvance Viral reagents also showed consistent Ct values comparison to RNA Standard (SARS-CoV-2 Standard without extraction). RNAdvance Viral reagents demonstrate a greater than 95% recovery efficiency with no PCR inhibition. The recovery efficiency was calculated by Ct of RNAdvance Viral/Ct of RNA Standard for Primer SARS-CoV-2 N1, SARS-CoV-2 N2, and SARS-CoV-2 N3 at all viral titers.

The analytical performance is determined as the lowest viral copy concentration that shows PCR amplification. For both extractions, 0.2 copies/μL of Exact Diagnostics RNA Standard showed undetermined Ct, indicating analytical performance is between 0.2-2 copies/μL.

Sample Name SARS-CoV-2 N1 SARS-CoV-2 N2 SARS-CoV-2 N3 RP
Competitor A_200 28.550 28.572 27.923
26.353
Competitor A_20 32.407 32.392 31.294 26.473
Competitor A_2 35.851 35.978
35.713
26.615
Competitor A_0.2 Undetermined Undetermined
Undetermined
26.646
RNAdvance Viral_200 28.775 28.633
27.891 26.346
RNAdvance Viral_20
31.971 32.082
31.252 26.382
RNAdvance Viral_2
35.126 35.680
35.979
26.409
RNAdvance Viral_0.2
40.3239 Undetermined
38.065 26.364
RNA Standard_200 27.676 28.147
27.201
27.976
RNA Standard_20
31.178 31.530 30.527
31.472
RNA Standard_2 35.491 34.925
35.033
35.102
RNA Standard_0.2
Undetermined 37.801 36.253
37.458

Table 1. Extraction proficiency evaluation

2. Confirmation of Analytical Performance

To confirm the analytical performance (1 copy/μL) obtained from the estimation step for RNAdvance Viral reagents, RNA was extracted from Exact Diagnostics RNA Standard and spiked into the pooled negative universal transport media. The input concentration of RNA Standard is 1 and 2 copies/μL. Quadruplicate samples were run in each input concentration.

Ct value was assessed via qRT-PCR using the protocol in the Assay Set Up section described in the CDC 2019 Novel Coronavirus (2019-nCoV) RT-PCR Diagnostic Panel instructions (CDC-006-00019). For each sample, N1, N2, N3, and RP genes were tested. Additionally, RUO controls, 2019-nCoV_N_Positive Control (IDT: 10006625) and qPCR negative controls (NTC Ambion, cat# AM4932), were included to ensure proper testing process.

Both 1 and 2 copy/μL of RNA Standard show comparable Ct value (Table 2) and confirmed that 1 copy/μL is the analytical performance for RNAdvance Viral reagents.

Sample Name SARS-CoV-2 N1 SARS-CoV-2 N2 SARS-CoV-2 N3 RP
RNAdvance Viral_2-1
 34.443 34.892 33.656
26.408
RNAdvance Viral_2-2
 34.933 35.403 34.420 26.579
RNAdvance Viral_2-3
 34.627 35.489
34.272
26.405
RNAdvance Viral_2-4
 35.304 34.912
34.493
26.697
RNAdvance Viral_1-1
 34.314 35.901
34.785 27.612
RNAdvance Viral_1-2
 35.781 36.520
36.592 27.659
RNAdvance Viral_1-3
 34.966 36.637
34.827
27.611
RNAdvance Viral_1-4
 35.550 34.625
35.730 27.757

Table 2. RNAdvance Viral reagents Analytical Performance confirmation

3. Sample Concordance

Sample concordance was demonstrated comparing Ct values on previously tested RNA samples extracted using Competitor A. There were 24 known positive with Ct values <40, and 23 known negative (Table 3) SARS-CoV-2. All samples were collected with a COPAN swab and stored in the HEALTHLINK universal transport media before extraction. RNA from all of the samples was extracted using RNAdvance Viral reagents manually. RT-PCR assay was performed with N2 and RP primer/probe sets. 2019-nCoV_N_Positive Control (IDT: 10006625) and qPCR negative control (NTC Ambion, cat# AM4932) were included to ensure a proper testing procedure.

The data indicates 100% concordance for all positive samples. However, 1 of 23 previously negative samples was positive, indicating 96% concordance. The operator noted it as a sample handling error.

Sample Type Sample Number Ct <40 No./Total No. Concordance No./Total No.
Known Positive  24 24/24 24/24
Known Negative  23 1/23 22/23

Table 3. Ct Values and comparative performance of known positive and negative SARS-CoV-2 samples

Conclusion

This evaluation demonstrates a research use extraction solution for reliable isolation of viral RNA from swab samples collected in universal transport media for research. The data presented shows RNAdvance Viral extraction efficiency is greater than 95% and does not inhibit downstream qRT-PCR analysis. The analytical performance is as low as 1 copy/μL viral RNA. The detection concordance demonstrated with the known positive and negative samples is 100% and 96%, respectively, and an accuracy of about 98% compared to RNA extracted with a competitor kit Ct values. The evaluation data produced has enabled the use of the RNAdvance Viral reagents for viral RNA extraction from swab sample in universal transport media. 

 

1Qi Wei, Ph.D.; 1Weiming Shen, MS, ASCPCM; 1Haixue Gan; 1Dianelis Mondejar Alvarez; 1Mendizabal; 1Tania E. Pumariega; 2Cindy Heath; 2Han Wei, Ph.D.
Affiliation: 1Miami Cancer Institute, Miami, FL; 2Beckman Coulter Life Sciences, Indianapolis, IN

Beckman Coulter Life Sciences and Miami Cancer Institute

 

Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only, and is not validated by Beckman Coulter.

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Miami Cancer Institute
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 Miami Cancer Institute Conducted Virology Research Using RNAdvance Viral 

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Application Note
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