RNA Sample Preparation

A Rapid, Simple, and Low-Cost RNA Sample Preparation Method
Based on Chelex™ 100 Resin

Elizabeth Dreskin,1 Angelica Olcott,1 and Ertan Ozyamak1
1Bio-Rad Laboratories, Inc., 6000 Alfred Nobel Drive, Hercules, CA 94547, USAAbstract

Chelex 100 Resin is a heat-stable bead polymer that captures divalent cations, such as Mg2+ andCa2+, from solutions. This property allows the inhibition of nucleases in biological samples, thus
preserving DNA or RNA molecules for downstream detection. Here, rapid protocols using Chelex 100Resin and a related product, InstaGene Matrix, were compared against a commercial RNA extraction
kit for sample preparation from a variety of sample types for use in reverse transcription quantitative
PCR (RT-qPCR) testing.
Introduction
Frequent ongoing surveillance is an effective strategy to minimize
or prevent community spread of infectious diseases. However,
several factors such as high cost and insufficent throughput
can hamper such public health efforts. To aid in community
surveillance studies, a low-cost, rapid, and simple protocol for
RNA sample preparation based on Chelex 100 Resin, a chelatingresin, was devised. Treatment with Chelex 100 Resin captures
divalent cations, inhibiting nucleases in biological samples.
Chelex 100 Resin is a bulk resin, which can be prepared at theconcentration or percentage of the user’s choice, and is a productoften used for higher throughput needs. For standard laboratory

1Add Chelex 100 Resinsuspension to sample
and mix.

2

Heat sample;
e.g., at 95°C for
2–10 min.

3

applications, Chelex 100 Resin is available as InstaGene Matrix, asmall volume (20 ml), ready-made 6% Chelex 100 Resin suspension.
Either Chelex 100 Resin or InstaGene Matrix can be used to prepare
RNA samples simply by mixing the product with the sample andperforming a short heat treatment (Figure 1). The supernatant
can then be used directly for PCR, RT-qPCR, reverse transcription
Droplet Digital™ PCR (RT-ddPCR), or reverse transcription
loop-mediated isothermal amplification (RT-LAMP).
Our data show that a range of sample types prepared with
Chelex 100 Resin or InstaGene Matrix yield amplifiable RNA andgenomic DNA comparable to that obtained using a commercial
extraction kit at a fraction of the cost and preparation time.

Separate Chelex 100 beads
from sample by centrifugation,
settling, or filtration.

4Use supernatant in downstream
step, such as PCR, RT-qPCR,RT-ddPCR, or RT-LAMP.

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NovemberFig. 1. Chelex 100 Resin–based sample preparation workflow overview. RT-qPCR, reverse transcription quantitative PCR; RT-ddPCR, reverse transcription
Droplet Digital PCR; RT-LAMP, reverse transcription loop-mediated isothermal amplification.

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A Rapid, Simple, and Low-Cost RNA Sample Preparation Method Based on Chelex 100 Resin

For Chelex 100 Resin and InstaGene Matrix, the final sample
volume was 100 μl, whereas samples extracted using theQIAamp Kit were eluted to a final volume of 50 μl. Input sample
volume was kept consistent for all methods (see previousparagraph). Quantification cycle (Cq) values were adjusted
to reflect the differences in sample volume.*

Materials and Methods
Samples of A549 cells (adenocarcinomic human alveolar basal
epithelial cells), cheek buccal cells, and saliva were prepared inVirus Transport Media (VTM) (Neuromics, catalog #VTM-4) thatwas spiked with the synthetic RNA template at 1/10 of the final
volume. Cheek buccal cells were isolated from the inside cheekarea by scraping with a sterile pipet tip. Saliva samples wereobtained by direct transfer into a standard laboratory tube. For
viral RNA detection tests, influenza virus A was used at 2x, 4x,
and 6x the previously determined limit of detection (LOD).

For RNA stability tests, sample aliquots were stored at 4°C after
preparation with InstaGene Matrix or a QIAamp Kit as describedabove. Sub-aliquots were removed to a plate with RT-qPCR mastermix at 1, 2, 3, and 4 hr timepoints, then evaluated by RT-qPCR todetermine RNA integrity over time.

Chelex 100 Resin, molecular biology grade (Bio-Rad Laboratories,Inc. #1421253), was prepared at a 6% w/v suspension in Tris-EDTA(TE) buffer (pH 8.0) or double distilled water (ddH2O), followed bythree washes of the same before being brought to final volume in
TE buffer or water. Chelex 100 Resin in ddH2O was used after initial
testing because TE buffer showed similar results. InstaGene Matrix,20 ml (Bio-Rad, #7326030) is a ready-made qualified 6% suspensionof Chelex 100 Resin. Fifty microliters of either suspension wasadded to each 50 μl sample. The bead suspensions were vortexed
after every three dispenses to keep the resin beads from settlingprior to pipetting. For convenience, 8-well PCR strip tubes wereused. Once combined, the Chelex 100 Resin and InstaGene Matrix
samples were placed in a thermal cycler and incubated at 95°C
for 10 min. Samples were cooled to 4°C, centrifuged briefly forseveral seconds in a microcentrifuge to sediment the beads, andthen 50 μl of supernatant was transferred into a fresh tube, making
sure to avoid disturbing the sedimented beads. To compare
results obtained using Chelex 100 Resin and InstaGene Matrix to acommercial kit, the QIAamp Viral RNA Mini Kit (QIAGEN, #52904)
was used according to the manufacturer’s instructions with 50 μlof sample per replicate. Nucleic acids were eluted off the column
such that the final volume for each replicate was 50 μl.

*To account for the higher dilution of the sample, the obtained Cq values for
Chelex 100 Resin and InstaGene Matrix were manually decreased by 0.85 cycles.
This value was determined by estimating the difference in final sample volume whenusing the resin-based methods versus the kit-based method. We confirmed theuse of Cq adjustment across several samples and it was equivalent to bringing
the recovered sample volumes to the same level.

Results
RNA Preparation from Cultured Cells

Our data demonstrate that combining cell suspensions with
Chelex 100 Resin or InstaGene Matrix and heating at 95°C
lyses cells and yields amplifiable RNA, genomic DNA, andspike-in RNA (when used). This shows that Chelex 100 Resinand InstaGene Matrix work effectively to chelate out divalentcations that may promote nucleic acid degradation or interferewith reverse transcription or DNA polymerase activities.
Data from 500 cell and 5,000 cell inputs are shown in Figure 2.
From these results one can conclude that Chelex 100 Resin andInstaGene Matrix perform similarly to each other and that thepreparation method is suitable for downstream analysis of both
RNA and DNA. Additionally, both products perform comparably
to the QIAGEN QIAamp Kit.

RT-qPCR was carried out using Reliance One-Step Multiplex
RT-qPCR Supermix and several different assays run on a
Bio-Rad real-time thermal cycler. To detect genomic DNA (gDNA),the RNase P Assay (Cy5.5; Bio-Rad, #12004601) was used.
The PrimePCR Reverse Transcription Control Probe Assay(FAM; Bio-Rad, assay ID: qHsaCtlP0001001; referred to hereafteras the PrimePCR RT Control Assay) was used to detect spike-inRNA, and the PrimePCR Probe Assay: ACTB (HEX; Bio-Rad,assay ID: qHsaCEP0036280) was used to detect endogenous
RNA. Influenza A was detected using a proprietary probe assay
designed against the M1 gene. Reactions (20 μl each) were setup using 10 μl of master mix and 10 μl of template. Spike-inexperiments used the synthetic RNA template that ships with thePrimePCR RT Control Assay. The following protocol was used
for RT-qPCR thermal cycling: 50°C for 10 min, 95°C for 10 min,followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec.
For all experiments, three replicate preparations were made,and each preparation was analyzed by RT-qPCR in triplicate.

© 2022 Bio-Rad Laboratories, Inc.

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A Rapid, Simple, and Low-Cost RNA Sample Preparation Method Based on Chelex 100 ResinAA
32403035

Average Cq

Average Cq

2826243025222020
Chelex 100 Resin

InstaGene Matrix

QIAGEN QIAamp Viral RNA
Mini Kit

15Sample preparation methodB

5505005,000
Input sample concentration, number of cells/50 μl

50,0005505005,000
Input sample concentration, number of cells/50 μl

50,0005505005,000
Input sample concentration, number of cells/50 μl50,000

B3040283826

36
Average Cq

Average Cq

32242234323020
Chelex 100 Resin

InstaGene Matrix

QIAGEN QIAamp Viral RNAMini Kit

28Sample preparation method

26Fig. 2. Comparison of Chelex 100 Resin, InstaGene Matrix, and QIAGEN
QIAamp Viral RNA Mini Kit for extraction of genomic DNA and spike-in RNA.
Comparison of average Cq values for genomic DNA ( ) and the spike-in RNA
control ( ) for samples prepared with Chelex 100 Resin, InstaGene Matrix,or a QIAGEN QIAamp Kit. Each starting sample consisted of: A, 500 A549 cells;
B, 5,000 A549 cells. Results are averages of three samples per treatment, eachassayed by reverse transcription quantitative PCR (RT-qPCR) in technical triplicate.
Error bars indicate standard deviation. The average Cq values for spike-in RNA (—)
and genomic DNA (—) prepared using the QIAamp Kit are marked for reference.
Cq, quantification cycle.

C4240
38
36
Average Cq

34

Chelex 100 Resin and InstaGene Matrix perform almost as wellas the QIAamp Kit for spike-in reverse transcription (RT) control
RNA, genomic DNA, and endogenous RNA over a wide rangeof cell concentrations (from 5 to 50,000 cells per 50 μl) as shownin Figure 3. Each data point shows the averaged results of threesamples per treatment, each assayed in three technical replicatesby RT-qPCR, yielding a total of nine measurements for each sample.

3230
2826
242220Fig. 3. Comparison of nucleic acid extraction methods at varying cell input
ranges. Chelex 100 Resin and InstaGene Matrix work well for sample preparation
over a wide range of input A549 cell numbers. Graphs show average Cq values
obtained for endogenous RNA (A), spike-in RNA (B), and genomic DNA (C) samples
after preparation with InstaGene Matrix ( ), Chelex 100 Resin ( ), or a QIAGEN
QIAamp Viral RNA Mini Kit ( ). Error bars indicate standard deviation.
Cq, quantification cycle.

© 2022 Bio-Rad Laboratories, Inc.

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35

Stability of Prepared Samples

The stability of the prepared samples at 4°C for up to 4 hours wastested to determine whether this method could potentially workfor sample preparation in assays that have specific sample typerequirements. The data presented in Figure 4 indicate that samples
prepared with InstaGene Matrix over a range of input cell counts
are stable. InstaGene Matrix and Chelex 100 Resin were shown tobe equivalent in terms of the stability of extracted samples at 4°C(data not shown).

Average Cq

34

33

32

31

2.5
2.0

30

1.5

2 x LOD

Average ∆Cq

1.0

4 x LOD
Sample preparation method

6 x LOD

Fig. 5. InstaGene Matrix is effective at sample preparation for viral RNA
detection. Average Cq values obtained from reverse transcription quantitative
PCR (RT-qPCR) analysis of samples containing influenza A viral RNA. Samples wereprepared using InstaGene Matrix ( ) or a QIAGEN QIAamp Viral RNA Mini Kit ( ).
Cq, quantification cycle; LOD, limit of detection.

0.5
0.0
–0.5
–1.0
–1.5

Table 1. Comparison of Cq values obtained for detection of influenza Ain prepared viral RNA samples using InstaGene Matrix versus a QIAGEN
QIAamp Viral RNA Mini Kit.

–2.0
–2.5
550
500
5,000Input sample concentration, number of cells/50 μl50,000Fig. 4. RNA stability of samples using InstaGene Matrix. Samples prepared with
InstaGene Matrix are stable at 4°C for at least 4 hours postpreparation. The averagechange in quantification cycle (ΔCq) values between prepared samples added to
RT-qPCR master mix immediately and samples added after being stored at 4°C for
4 hr is plotted on the y axis against the number of input A549 cells. Average ∆Cqvalues are shown for endogenous RNA ( ), genomic DNA ( ), and spike-in RNA ( ),and a boundary of ±1.0 ∆Cq is marked for reference (—).

InstaGene Matrix
Average CqSDAverage ∆Cq

2 × LOD

34.60.261

33.4

0.387

1.14 × LOD

33.90.252

32.3

0.307

1.5

6 × LOD

33.70.406

32.4

0.211

1.2LOD, limit of detection; Cq, quantification cycle; SD, standard deviation; ∆Cq, differencein Cq values.

For epithelial cells isolated from cheek buccal scrapes and saliva
samples, treatment with InstaGene Matrix could enable detectionof both spike-in RT control and genomic DNA (data not shown).
Owing to the presence of known PCR inhibitors and RNAses,RNA levels are expected to be lower in saliva samples, and theresulting Cq levels higher, compared to other sample types suchas epithelial cells from cheek buccal scrapes (Ostheim et al. 2020).

Preparation of Other Sample Types

The Chelex 100 Resin–based method was evaluated for sample
preparation of RNA and DNA from influenza A virus (Figure 5,Table 1), as well as cheek scrapes and saliva (data not shown).
For influenza A samples, template was detectable by RT-qPCR inChelex 100 Resin–treated samples even when input quantities as
low as twice the LOD were used, indicating that this is a suitable
method for quick and efficient preparation of virus samples.

© 2022 Bio-Rad Laboratories, Inc.

QIAGEN QIAamp Kit
Viral RNASD
Input Quantity Average Cq

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A Rapid, Simple, and Low-Cost RNA Sample Preparation Method Based on Chelex 100 Resin

Factoring in only the reagent cost, we estimate savings in the rangeof 18- to 36-fold per sample when InstaGene Matrix is used as
described in this protocol compared with RNA extraction kits frommajor manufacturers, as of the time of this publication. Further
significant savings can be achieved with Chelex 100 Resin.

Workflow Time, Cost, and Plastic Usage

A major benefit of the Chelex 100 Resin–based methods is theircost effectiveness. Researchers reported an estimated eightfold
cost reduction for SARS-CoV-2 detection when using Chelex 100
Resin–based methods, including the cost of collection tubes
and additional reagents (Guan et al. 2021). Another study indicatedthat the cost of Chelex 100 Resin required per sample was
50- to 170-fold cheaper than the per sample cost for traditional
DNA extraction kits or reagents (Lienhard and Schäffer 2021).

Sample preparation time

In addition to the reduced cost, the workflow enabled by Chelex
100 Resin or InstaGene Matrix also offers substantial reductions inboth the time required for sample preparation and the generationof biohazardous and plastic waste (Figure 6).

Cost

Waste generation

Application
RT-qPCRRNA extraction kits

RT-ddPCR

RT-LAMPRT-qPCR

Chelex 100 Resin

RT-ddPCR

RT-LAMP

Fig. 6. Comparison of sample preparation time, cost, plastic waste generation, and application suitability for Chelex 100 Resin–based methods versus
a commercial-grade kit for RNA or DNA isolation. RT-qPCR, reverse transcription quantitative PCR; RT-ddPCR, reverse transcription Droplet Digital PCR; RT-LAMP,
reverse transcription loop-mediated isothermal amplification.

sample preparation from mammalian cells and viruses: the cost
per preparation is significantly lower than available RNA extraction
kits, the protocol is rapid, and much less plastic waste is generated
per preparation. In cases where a larger number of samples will be
run consistently, Chelex 100 Resin may be a preferred option due
to bulk pricing advantages.

As a result, the use of Chelex 100 Resin or InstaGene Matrix canstreamline a given molecular detection workflow and significantly
reduce costs with the added benefits of excellent sustainability due
to far lower waste generation. Chelex 100 Resin–based sample
preparation methods are suitable for RT-qPCR (shown here;
Ulloa et al. 2020), RT-ddPCR (Guan et al. 2021), and RT-LAMP(Yaren et al. 2021).

The additional advantage of Chelex 100 Resin is the ability tocustomize the suspension buffer as well as the suspension
concentration. In this study, we chose a 6% (w/v) Chelex 100 Resinsuspension, however, suspensions of 10–20% are typically usedin sample preparation protocols and suspensions of up to 50%
have been reported. A higher Chelex 100 Resin concentration may
yield improved results in cases where a higher capacity to capture
divalent ions, and therefore to limit nuclease activity, is needed.
Overall, for community surveillance purposes, Chelex 100 Resinand InstaGene Matrix offer compelling advantages over othercommercial kits, especially when availability of supplies is limited.

Conclusions
Commercial DNA and RNA extraction kits isolate nucleic acids
at high enough purity and yield for use in molecular assays andare considered the gold standard for detection by RT-qPCR.
However, certain applications may not justify the high cost andslow processing speed (low throughput) associated with suchkits. Here we demonstrated that both Chelex 100 Resin andInstaGene Matrix, which stabilize RNA/DNA in samples throughchelation of divalent cations, provided results highly comparable toa commercial RNA extraction kit from QIAGEN. InstaGene Matrix
and Chelex 100 Resin offer several substantial benefits for RNA© 2022 Bio-Rad Laboratories, Inc.

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It should be pointed out that because the Chelex 100 Resin–based protocols do not isolate nucleic acids, the reagents usedin RT-qPCR may play a critical role in the detection of the RNA
or DNA targets queried. We have found that Reliance One-Step
Multiplex RT-qPCR Supermix can dependably detect these targets
when the sample accounts for 50% of the final reaction volume.
Because this reagent is supplied as a 4x solution, it is theoreticallypossible to add sample volumes larger than 50% of the final
reaction volume; however, this condition was not tested here.
This method is potentially automatable using a liquid handler
with an on-deck heating block or a thermal cycler that can handle
96-well and 384-well plates as well as microfuge tubes. Therefore,
this method may also enable higher throughput screening.

Ordering Information
Catalog #Description

1421253

Chelex 100 Chelating Resin, molecular biology grade, 200–400 mesh,sodium form, 50 g
InstaGene Matrix, 20 ml7326030

Visit bio-rad.com/Chelex100-RNA for more information and a listof publications using Chelex 100 Resin.
BIO-RAD, CHELEX, and DROPLET DIGITAL are trademarks of Bio-Rad Laboratories, Inc. incertain jurisdictions. All trademarks used herein are the property of their respective owner.

References
Guan B et al. (2021). Sensitive extraction-free SARS-CoV-2 RNA virus detection using
a chelating resin. iScience 24, 102960.
Lienhard A and Schäffer S (2019). Extracting the invisible: Obtaining high quality DNA
is a challenging task in small arthropods. PeerJ 7, e6753.
Ostheim P et al. (2020). Overcoming challenges in human saliva gene expression
measurements. Sci Rep 10, 11147.
Ulloa S et al. (2020). A simple method for SARS-CoV-2 detection by rRT-PCR without
the use of a commercial RNA extraction kit. J Virol Methods 285, 113960.
Yaren O et al. (2021). Ultra-rapid detection of SARS-CoV-2 in public workspaceenvironments. PLoS One 16, e0240524.

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