Journal of Microbiological Methods 144 (2018) 1–7

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Journal of Microbiological Methods

journal homepage: www.elsevier.com/locate/jmicmeth

Have you tried spermine? A rapid and cost-effective method to eliminate MARK
dextran sodium sulfate inhibition of PCR and RT-PCR
Łukasz Krycha,⁎, Witold Kotb, Katja M.B. Bendtsenc, Axel K. Hansenc, Finn K. Vogensena,
Dennis S. Nielsena
a
Department of Food Science, Faculty of Science, University of Copenhagen, Denmark
b
Department of Environmental Science, Aarhus University, Denmark
c
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

A R T I C L E I N F O A B S T R A C T

Keywords: The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human in-
Spermine flammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated
Dextran sodium sulfate with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and
DSS reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective
Quantitative reverse transcription PCR
protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an ef-
RT-qPCR
DSS inhibition
fective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that
spermine could be used to counteract DSS inhibition of PCR and RT.
We investigated the means of adding spermine in an adequate concentration to PCR based protocols (in-
cluding qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition.
Within the range up to 0.01 g/L, spermine can be added to PCR/qPCR or RT prophylactically without a sig-
nificant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08 g/L can be used to
recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32 g/L. For optimal quantitative
analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric
based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed
to an unknown concentration of DSS.
In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-
step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative
or quantitative character of the analysis.

1. Introduction
In 1985 Toshifumi Ohkusa introduced administration of the he-
parin-like sulfated polysaccharide Dextran Sulfate Sodium (DSS) as a
method for induction of colitis in animal models (Ohkusa, 1985). DSS
interrupts the mucosal barrier of mainly the colon, producing clinical
and morphological traits of human inflammatory bowel disease (IBD) in
a concentration-, duration-, and frequency-dependent manner. The
model of IBD is widely used due to ease of induction, controllability,
and translational value (Perše and Cerar, 2012). Nonetheless, the exact
mechanism of DSS induced inflammation in the colon remains un-
known. Several hypotheses have been proposed, including direct toxic
effects on the epithelium, altered macrophage function, autoimmune-
like reactions between intestinal lymphocytes and epithelial cells, and
activation of different T-cell subsets (Dieleman et al., 1998; Ni et al.,
1996; Okayasu et al., 1990). DSS is soluble in water and most com-
monly administered in the drinking water of rodents at concentrations
ranging from 1 to 8%. The severity and characteristics of colitis depends
on the molecular weight (Kitajima et al., 2000), sulfur content (Bamba
et al., 2012), and dosage per animal body weight (Vowinkel et al.,
2004), therefore these three parameters should be reported in studies
exploiting DSS. Furthermore, the susceptibility to DSS is highly de-
pendent on genetic background, gender, gut microbiota composition,
and environment (Perše and Cerar, 2012). The importance of gut mi-
crobiota in the onset, progression and severity of pathological condi-
tions in the gastrointestinal tract, including IBD, is a subject of intensive

Abbreviations: ANOSIM, analysis of similarities; Ct, threshold cycle; DSS, Dextran Sulfate Sodium; IBD, inflammatory bowel disease; OTU, Operational Taxonomic Unit; PCoA, Principal
Coordinate Analysis; RFU, Relative Fluorescence Unit; RT, reverse transcription
⁎
Corresponding author.
E-mail address: krych@life.ku.dk (Ł. Krych).

http://dx.doi.org/10.1016/j.mimet.2017.10.015
Received 9 June 2017; Received in revised form 27 October 2017; Accepted 27 October 2017
Available online 28 October 2017
0167-7012/ © 2017 Elsevier B.V. All rights reserved.
Ł. Krych et al.
research in both humans and animal models and has been widely re-
viewed (Seksik et al., 2006; Shanahan and Quigley, 2014). An altered
balance of beneficial and harmful bacteria, termed dysbiosis, can tip the
development of gastrointestinal disease depending on the host en-
vironment (Hansen et al., 2012; Larsen et al., 2009; Wen et al., 2008).
Therefore, the relevance of knowing the bacterial population in the gut
of animal models in this context is evident. Many state of the art high
throughput molecular techniques used for monitoring microbial com-
munities relay on PCR.
DSS has previously been shown to inhibit Taq polymerase activity in
a dose-dependent manner causing a strong inhibition of PCR above the
concentration of 0.01 g/L (Kerr et al., 2012; Viennois et al., 2013). The
inhibition of reverse transcription (RT) was shown not to be dosage-
dependent, yet leads to significant, but not full inhibition above a DSS
concentration of 5.10− 4 g/L. Two purification methods for RNA have
been proposed to remove DSS from RNA samples (Kerr et al., 2012;
Viennois et al., 2013). The Poly-A-mediated mRNA purification method
proposed by Kerr et al. although efficient, is restricted to eukaryotic
samples (Kerr et al., 2012). Lithium chloride based purification seems
to be more universal, however additional purification can affect the
quality of RNA and consequently the quantitative analysis (Viennois
et al., 2013). Finally, neither of the two methods can be applied to
remove DSS from DNA or cDNA samples. Accumulation of DSS in the
gastro-intestinal tract of colitis model animals can severely limit the
possibility of DNA based analysis of the gut microbiota with PCR.
Especially, if one seeks to investigate the time point during or shortly
after the exposition to DSS when its concentration is often high enough
to cause full inhibition of PCR.
In 1971 Ray Wu used spermine and polylysine to remove inhibition
of polynucleotide kinase caused by a low concentration of poly-
saccharide sulfates (Wu, 1971). Spermine is a tetra-cationic amine
(Spm4 +) and thanks to its highly positive charge it binds to the phos-
phate groups of the DNA molecule. Korolev et al. demonstrated that the
long and flexible spermine molecule forms bridges linking the phos-
phate groups between neighbouring DNA duplexes and displays a high
presence in the minor groove of the DNA (Korolev et al., 2002). Al-
though the mechanism of spermine anti-inhibitory properties against
DSS remains unclear, it seems reasonable to assume that positively
charged spermine interacts with negatively charged sulfate in the DSS
molecule.
We hypothesized that spermine has higher affinity to DSS than to
DNA and therefore when administrated in equilibrated proportions it
binds most of DSS molecules preventing PCR inhibition. We demon-
strate herein the optimal proportions of spermine that can be applied in
order to counteract the inhibition of PCR and two-step RT-qPCR caused
by a given concentration of DSS in environmental samples.
2. Materials and methods
2.1. Spermine and dextran sulfate sodium salt
Spermine (Sigma-Aldrich, MO, USA, S3256), and dextran sulfate
sodium (DSS, cat. no. 160110, lot no. M2709, MW = 36,000–50,000,
MP Biomedicals, USA) were dissolved in Milli-q water (Millipore) and
stored at 2–8 °C according to users' manual.
2.2. Nucleic acids extraction
Cellular DNA used for PCR, qPCR, and 16S rRNA gene amplicon
sequencing was extracted from mouse fecal samples using PowerSoil
DNA Isolation Kit (MoBio Laboratories, CA, USA) according to the
manufacturer's instructions including an initial bead-beating step using
FastPrep-24™ 5G (MP Biomedicals, CA, USA).
Total RNA was extracted from murine intestinal tissue with
chloroform (Merck, VWR, Herlev, Denmark) and ethanol (Kementyl,
VWR), and subsequently isolated using the RNeasy Lipid kit (Qiagen)
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Journal of Microbiological Methods 144 (2018) 1–7
including on-column digestion of DNA using the RNase-Free DNAse set
(Qiagen) according to manufacturer's protocol. Total RNA concentra-
tion and purity was measured using a NanoDrop ND-1000 spectro-
photometer (Saveen and Werner AB, Sweden) and the RNA integrity
assessed using the Agilent Bioanalyzer 2100 and RNA 6000 Nano Kit
(Agilent Technologies).
2.3. PCR inhibition test
Standard PCR targeting the V3 region of the 16S rRNA gene was
used with following primers: 338_F: 5′-CCTACGGGWGGCAGCAG-3′
and 518_R: 5′-ATTACCGCGGCTGCTGG-3′ (Integrated DNA
Technologies; Leuven, Belgium). PCR reactions containing 12 μL of
DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, CA, USA),
0.5 μL of each primer (5 μM), 5 μL of genomic DNA (~ 20 ng/μL), 2 μL
of either spermine, DSS, or both (in respective concentrations), and
nuclease-free water to a total volume of 20 μL, were run on a SureCycler
8800 (Agilent, CA, USA). Applied cycling conditions were:
Denaturation at 95 °C for 2 min; 33 cycles of 95 °C for 15 s, 60 °C for
15 s and 72 °C for 30 s; followed by final elongation at 72 °C for 5 min.
PCR products were visualised on 1.5% agarose gels.
qPCR targeting the same region was conducted on a 7500 Fast Real-
time PCR System (Applied Biosystems, USA, CA). The reaction mix
composed of 12 μL of FastStart Universal SYBR Green Master (Roche,
Basel, Switzerland), 0.5 μL of each primer (5 μM), 5 μL of genomic DNA
(~ 20 ng/μL), 2 μL of either spermine, DSS, or both (in respective
concentrations), and nuclease-free water to a total volume of 20 μL. The
thermal profile was set as follows: Denaturation at 95 °C for 10 min;
40 cycles of 95 °C for 15 s, and 60 °C for 60 s; melting step from 60 °C to
95 with 1% heating speed. Each sample was prepared in 6 replicates
and shifts in threshold cycle (Ct) average values tested with Student's t-
test.
2.4. Tag-encoded Illumina high throughput amplicon sequencing
DNA originating from mouse fecal samples were exposed to varying
spermine, DSS, or spermine/DSS concentrations and afterwards their
compositions were determined using tag-encoded 16S rRNA gene
MiSeq-based (Illumina, CA, USA) high throughput sequencing. Samples
were analysed in 8 technical replicates. Three samples (2 controls and 1
spermine 0.02 g/L) were excluded due to the low reads number.
The V3 region (~ 190 bp) of the 16S rRNA gene was amplified using
primers compatible with Nextera Index Kit (Illumina): NXt_388_F: 5′-
TCGTCGGCAG CGTCAGATGT GTATAAGAGA CAGACWCCTA
CGGGWGGCAG CAG-3′ and NXt_518_R: 5′-GTCTCGTGGGC
TCGGAGATGTG TATAAGAGAC AGATTACCGC GGCTGCTGG-3′
(Integrated DNA Technologies; Leuven, Belgium). PCR reactions con-
taining 12 μL AccuPrime™ SuperMix II (Life Technologies, CA, USA),
0.5 μL of each primer (10 μM), 5 μL of genomic DNA (~ 20 ng/μL), 2 μL
of either spermine, DSS, or both (in respective concentrations), and
nuclease-free water to a total volume of 20 μL were run on a SureCycler
8800. Cycling conditions applied were as follows: 95 °C for 2 min;
33 cycles of 95 °C for 15 s, 55 °C for 15 s and 68 °C for 30 s; followed by
final step at 68 °C for 5 min. To incorporate primers with adapters and
indexes, PCR reactions contained 12 μL Phusion High-Fidelity PCR
Master Mix (Thermo Fisher Scientific, MA, USA), 2 μL corresponding P5
and P7 primer (Nextera Index Kit), 2 μL PCR product and nuclease-free
water for a total volume of 25 μL. Cycling conditions were as follows:
98 °C for 1 min; 12 cycles of 98 °C for 10 s, 55 °C for 20 s and 72 °C for
20 s; and 72 °C for 5 min. The amplified fragments with adapters and
tags were purified using AMPure XP beads (Beckman Coulter Genomic,
CA, USA). Prior to library pooling clean constructs were quantified
using a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) and mixed
in approximately equal concentrations to ensure even representation of
reads per sample followed 250 bp pair-ended MiSeq (Illumina, CA,
USA) sequencing performed according to the instructions of the
Ł. Krych et al. Journal of Microbiological Methods 144 (2018) 1–7

manufacturer.
The raw dataset containing pair-ended reads with corresponding
quality scores were merged and trimmed using fastq_mergepairs and
fastq_filter scripts implemented in the UPARSE pipeline (Edgar, 2013).
The minimum overlap length of trimmed reads (150 bp) was set to
100 bp. The minimum length of merged reads was 150 bp. The max
expected error E = 2.0, and first truncating position with quality score
N ≤ 4. Purging the dataset from chimeric reads and constructing de
novo Operational Taxonomic Units (OTU) were conducted using the
UPARSE pipeline (Edgar, 2013). The green genes (13.8) 16S rRNA gene
collection was used as a reference database (McDonald et al., 2012).
Quantitative Insight Into Microbial Ecology (QIIME) open source soft-
ware package (1.7.0 and 1.8.0) was used for subsequent analysis steps
(Caporaso et al., 2010).
Principal coordinate analysis (PCoA) was conducted with the jack-
knifed beta diversity workflow based on 10 distance metrics calculated
using 10 subsampled OTU tables. The number of sequences taken for
each jackknifed subset was set to ~85% of the sequence number within
the most indigent sample (3000 reads/sample).
Analysis of similarities (ANOSIM) was used to evaluate group dif-
ferences using weighted and unweighted UniFrac (Lozupone and
Knight, 2005) distance matrices that were generated based on rarefied
(3000 reads/sample) OTU tables. The relative distribution of the genera
registered was calculated for unified and summarized in the genus level
OTU tables.
The G test of independence (q_test) and ANOVA determined re-
spectively: qualitative (presence/absence) and quantitative (relative
abundance) association of OTUs with tested categories. These were
calculated based on 1000 subsampled OTU-tables rarefied to an equal
number of reads (3000).

2.5. Reverse transcription inhibition test

RNA was treated with DNA-free™ DNA removal Kit (Life

Technologies, CA, USA). The RT-PCR was conducted using High
Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific)
according to the manufacturer's instructions. The RNA (before the RT
step) or cDNA exposed to varying concentrations of spermine, DSS or
both were tested with qPCR using reaction mix and the temperature
profile as described above. Primers used for this assays were targeting
ribosomal protein L13A (Rpl13a) as previously described (Rune et al.,
2013). Each sample was prepared in 6 replicates and shifts in Ct
average values tested with Student's t-test.
2.6. Fluorometric quantification of DNA and RNA
Varying concentrations of spermine and DSS were mixed with
10 ng/μL of DNA and measured using fluorescent based detection with
Qubit® dsDNA HS Assay Kit (Life Technologies, CA, USA). The mea-
surement was performed with Varioskan Flash Multimode Reader
(Thermo Fischer Scientific, MA, USA). Fluorescence was measured at
485/530 nm.
Varying concentrations of spermine and DSS were mixed with
10 ng/μL of RNA and measured using fluorescent based detection with
Qubit® RNA BR Assay Kit (Life Technologies, CA, USA). The measure-
ment was performed with Varioskan Flash Multimode Reader.
Fluorescence was measured at 630/680 nm.
Fig. 1. Spermine eliminates the DSS inhibition of PCR in at the optimal ratio 1:4.
The range of DSS concentration and corresponding spermine concentrations in which DSS
induced inhibition of PCR can be eliminated. Even inhibition caused by relatively high
concentration of DSS (0.32 g/L) can be eliminated by addition of spermine at ratio 4:1.
Reduced band intensity along increasing spermine concentrations indicates that excess of
unbound spermine influences the PCR efficiency.
ratio eliminating the PCR inhibition was 1:4. As an example, DSS in-
hibition up to a concentration of 0.32 g/L could be overcome by ad-
dition of 0.08 g/L of spermine (Fig. 1). In lower concentrations of DSS
ranging from 0.01 to 0.02 g/L the optimal spermine/DSS proportion
was 1:2. Analysis with qPCR showed that inhibition caused by DSS in
the concentration of 0.015 g/L could be counteracted by 0.0075 g/L of
spermine without affecting the reaction efficiency (Fig. 2). Further-
more, addition of spermine in concentrations between 0.0025 and
0.0075 g/L in the DSS-free assays seemed to improve the reaction ef-
ficiency shifting the Ct value of PCR for about one cycle less compared
to the control group. However, no differences between the technical
replicates exposed to different spermine concentrations could be ob-
served (Fig. 2).

3. Results
3.1. Spermine eliminates the DSS inhibition of PCR at the optimal ratio 1:4
Fecal derived bacterial DNA was amplified using universal primers
targeting the V3 region of the 16S rRNA gene. Spermine added to PCR
reactions eliminated the DSS inhibition in a dose dependent manner.
Above the DSS concentration of 0.02 g/L the optimal spermine/DSS
3.2. Excess of unbound spermine above the concentration of 0.1 g/L reduces
the efficiency of PCR
To test how the excess of unbound spermine in higher concentra-
tions affects PCR efficiency, unbalanced concentrations of spermine and
DSS ranging from 0.08 to 0.12 g/L were tested with qPCR. In the tested
range addition of spermine eliminated the inhibition of DSS at all sur-
veyed concentrations enabling the reaction. Increasing the

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Ł. Krych et al. Journal of Microbiological Methods 144 (2018) 1–7

Fig. 2. Fine-tuned spermine concentration counteracting

AVERAGE Ct SD Ct shift
spermine [g/L]

corresponding DSS inhibition in fecal derived bacterial

0.0000 7.8 0.3 0.0 DNA using qPCR.
0.0025 7.0 0.2 -0.8 Cycle threshold (Ct) values for technical replicates of fecal
derived bacterial DNA exposed to varying concentrations
0.0050 6.8 8.2 17.8 0.2 0.3 5.3 -1.0 0.4 10.1
of spermine, DSS, or both during the amplification.
0.0075 6.8 7.4 7.7 8.8 0.2 0.1 0.0 0.7 -0.9 -0.3 -0.1 1.0 Spermine at the concentration of 0.0025 g/L did not
0.00 0.01 0.015 0.02 0.00 0.01 0.015 0.02 0.00 0.01 0.015 0.02 counteract the DSS inhibition at any of the tested con-
DSS [g/L] centrations. Replicates in the red frames are significantly
different (t-test; p < 0.05) from the control set (no sper-
mine, nor DSS). Addition of 0.0075 g/L of spermine was the optimal concentration abolishing the DSS inhibition at the concentration of 0.015 g/L without affecting the Ct value.
Spermine in all tested concentrations in the absence of DSS has improved the qPCR efficiency by about 1 Ct. Black squares indicate no signal of qPCR.

concentration of DSS in this range did not change the qPCR efficiency,
while increasing the concentration of spermine clearly increased the Ct
values (Supplementary Fig. 1). Without presence of DSS the con-
centration of 0.08 g/L spermine, reduced the PCR efficiency sig-
nificantly, resulting in about 10 cycles higher Ct values, and at a con-
centration of 0.12 g/L with another 10 cycles (Supplementary Fig. 1).
Between the concentration of 0.12 g/L and 0.16 g/L spermine causes
absolute inhibition (data not shown). It is therefore clear that unbound
spermine in concentrations exceeding 0.01 g/L caused reduction in PCR
efficiency in a dose dependent manner. This can be also observed di-
rectly on the agarose gel through reduced band intensity along in-
creasing spermine concentrations (Fig. 1).
3.3. DSS interferes with library construction for amplicon sequencing. An
effect that can be counteracted using spermine
Technical replicates of 16S rRNA gene amplicon sequencing samples
exposed to varying concentrations of DSS and spermine, were used to
assess the influence of spermine excess (≥ 0.01 g/L) on the amplicon
sequencing. PCoA based on unweighted UniFrac distance matrices
showed no clear separation between tested categories (Fig. 3A). This
indicates that spermine administered in the tested concentration did not
significantly affect the qualitative analysis. Furthermore, testing with
the g-test of independence showed no significant differences between
the tested categories (data not shown).
Analysis using weighted UniFrac distances showed that bacterial
relative abundance was influenced by different concentrations of
spermine and/or DSS (Fig. 3B). However, the distances between sam-
ples within each cluster did not differ from distances between the
clusters (Supplementary Table 1). This indicates that exposure to the
same concentration of spermine affects the final amplicon profile ana-
logously making the beta diversity analysis comparable solely within
the category exposed to the same spermine concentration. ANOVA
analysis revealed moderate differences in the relative abundance of 14
out of 45 registered species between the categories exposed to various
spermine and/or DSS concentrations (Supplementary Fig. 2).
3.4. Spermine eliminates DSS inhibition of two-step RT-qPCR in a dose
dependent manner
DSS concentrations ranging from 0.04 g/L to 0.16 g/L spiked to
RNA prior RT were tested against concentrations of spermine ranging
from 0.01 g/L to 0.04 g/L added to cDNA using qPCR. Even very high
concentrations of DSS (0.16 g/L) could be counteracted by the addition
of spermine (0.04 g/L), though reducing the efficiency of the reaction
for about 1.5 Ct. Inhibition caused by DSS in the concentration of
0.04 g/L was counteracted by addition of spermine in all tested con-
centrations without significant shifts in Ct values. Combination of
0.08 g/L of DSS and 0.04 of spermine was the highest DSS concentra-
tion that could be counteracted without significant reduction in the PCR
efficiency. Spermine in all tested concentrations in the absence of DSS
did not cause significant reduction of the Ct value (Fig. 4).
Multiple attempts have been made to test the ease of spermine ad-
dition directly to RNA prior to RT. However, since the effect of sper-
mine on the DSS inhibition of RT was measured with qPCR, the com-
prehensive evaluation of the effect of various concertation ranges of
DSS and spermine was not feasible. This is mainly due to the fact that in
given concentrations of DSS/spermine it is impossible to distinguish
between the inhibition that took place during the RT and the carryover
inhibition during the qPCR. Diluting DSS containing cDNA to the

A B

Fig. 3. Influence of varying spermine concentrations on the qualitative and quantitative analysis in 16S rRNA gene amplicon sequencing.
PCoA plots based on unweighted (A) and weighted (B) UniFrac distance matrices calculated from rarefied (3000 reads/sample) OTU tables. No clear qualitative differences between the
categories were found within the unweighted UniFrac distances (ANOSIM R = 0.037; p = 0.198). Significant differences in bacterial relative distribution were recorded based on the
weighted UniFrac distances (ANOSIM R = 0.523; p = 0.001).

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Ł. Krych et al. Journal of Microbiological Methods 144 (2018) 1–7

Fig. 4. Spermine eliminates the DSS inhibition of two-step

AVERAGE Ct SD Ct shift
spermine [g/L]

RT-qPCR.
0.00 15.3 0.5 0.0 Cycle threshold (Ct) values for technical replicates of cDNA
0.01 15.3 15.7 16.7 32.7 0.1 0.2 0.3 5.2 0.0 0.4 1.5 17.4 exposed to varying concentrations of spermine, DSS, or
both during qPCR. Replicates in the red frames are sig-
0.02 15.4 15.6 16.4 17.2 0.1 0.1 0.3 0.3 0.1 0.3 1.1 1.9
nificantly different (t-test; p < 0.05) from the control set
0.04 15.7 15.4 15.6 16.8 0.1 0.1 0.1 0.8 0.4 0.1 0.3 1.5 (no spermine, nor DSS). Spermine added to the RT prior to
0.00 0.04 0.08 0.16 0.00 0.04 0.08 0.16 0.00 0.04 0.08 0.16 qPCR in the concentration of 0.04 g/L was the optimal
DSS [g/L] * amount abolishing the DSS inhibition at the concentration
of 0.08 g/L without affecting the Ct value. Even relatively
high concentration of DSS (0.16 g/L) was counteracted with 0.04 g/L of spermine with the reduction of reaction efficiency resulting in shift of 1.5 Ct value. Black squares indicate no
signal of qPCR. (*) DSS concentration is given for the RT reaction before the qPCR.

concertation below the inhibition threshold of PCR could give some
insight into the conditions during RT provided that concertation of
target cDNA was initially relatively high. In fact, a vague positive effect
of spermine on DSS inhibited RT could be observed when the cDNA was
diluted 10− 3 prior to qPCR (Supplementary Fig. 3). It was clear that
DSS caused only partial inhibition of the RT step and that presence of
spermine seemed to increase the efficiency of the reaction. Notably, the
difference between spermine containing samples and the control was
not significant due to higher variation in DSS spiked samples.
Furthermore, it cannot be unequivocally deducted to what extent and
which of the steps was facilitated by the addition of spermine: RT,
qPCR, or both. Importantly, in the concentration of 0.01 g/L spermine
did not affect the RT reaction neither in the DSS containing nor in the
DSS free samples indicating that in this concentration (0.01 g/L) sper-
mine could be added prophylactically directly to the RT step
(Supplementary Fig. 3).
3.5. Both spermine and DSS disrupt fluorometric quantification of DNA but
not RNA
Increasing concentrations of spermine in the range of 0.01 to
0.07 g/L disrupt fluorescence-based measurements in a dose dependent
manner. Between the concentrations of 0.01 and 0.04 g/L the signal
was dropping linearly whereas above the concentration of 0.04 g/L the
signal was reduced for about 30% for all remaining concentrations
(Fig. 5A). DSS administered in the concentrations ranging from 0.02 to
0.14 g/L reduced the readout about 40% in all tested concentrations
(Fig. 5A).
Combining spermine together with DSS reversed the signal strength
with the optimum (but not full) recovery at 1:4 ratio (Fig. 5B). Hence,
this observation can be applied for fine-tuning of a spermine con-
centration that optimally binds an unknown DSS concentration in en-
vironmental samples of DNA or cDNA.
Neither spermine nor DSS disrupted the fluorescence-based mea-
surements of RNA (data not shown). This indicates that fine tuning of
RNA samples using Qubit technology can be only performed after the
RT step on cDNA. However, as mentioned above even though this
method cannot be applied to assess the DSS concentration directly on
RNA samples, still low concentrations of spermine (below 0.01 g/L) can
be added prophylactically to the RT reaction without compromising its
efficiency (Supplementary Fig. 3).
4. Discussion
DSS induced colitis models are widely used in studies on in-
flammatory bowel disease mainly due to its simplicity (Perše and Cerar,
2012). DSS follows nucleic acids during the purification process and
causes strong inhibition of several downstream enzymatic reactions.
This includes full inhibition of PCR-based methods (when above 0.01 g/
L) and severe efficiency reduction of RT (Viennois et al., 2013). Dilution
of DSS contaminated samples would be the easiest approach to reduce
DSS concentration, however it will only work if the concentration of
nucleic acids is relatively high, while the DSS concentration relatively
low. In order to remove DSS surplus, two methods have been suggested,
yet they are limited to total RNA (lithium chloride) (Viennois et al.,
2013) or polyadenylated mRNA of eukaryotic origin (Kerr et al., 2012).
Furthermore, not much is known about the quality of RNA after using
these methods. To the best of our knowledge there is no easy and cost-
efficient method for removing DSS from DNA samples. Since DSS is the
most common compound used for studying colitis, Crohn's disease, and

A B

Fig. 5. Disruption of the fluorometric based quantification of DNA caused by spermine and DSS.
A) Reduction in the total DNA concentration expressed with relative fluorescence units (RFU) in the presence of increasing concentration of spermine (blue line) and DSS (red line). B)
Table presenting the level of signal recovery when both, spermine and DSS are administered with optimal recovery at ratio 1:4 (RFU = 8.1). (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

5
Ł. Krych et al.
ulcerative colitis there is a great need for a reliable and easy method to
counteract the inhibitory effect of DSS on DNA based downstream
analysis. Findings of the present study show that addition of the poly-
amine spermine can remarkably reduce the DSS inhibitory effect in
both PCR and RT-based methods. We demonstrated that 1:4 ratio of
spermine to DSS gives best performance. This fits with the fact that DSS
contains a negatively charged sulfate group while spermine is a tetra-
cationic polyamine (Spr4 +). Spermine added to the PCR or RT in
concentrations not exceeding 0.01 g/L was demonstrated not to reduce
reaction efficiency even in the absence of DSS, suggesting that within
this range it could be added prophylactically. The qPCR results indicate
that low concentrations of spermine (< 0.01 g/L) could even increase
the efficiency of reaction in DSS negative samples. One can speculate,
that this is due to spermine binding to other potential inhibitors present
in environmental samples.
The strong affinity of DSS to spermine molecules could also shed
light on the etiology of DSS induced colitis. Spermine is known to play
an important role in in metabolism of all eukaryotic cells and bacterial
proliferation (Gómez-Gallego et al., 2012). One could speculate that by
binding and deactivating cellular spermine DSS interrupts the essential
metabolism of epithelial cells causing their death leading to lesions.
Spermine was also shown to strongly interact with DNA (Feuerstein
et al., 1990; Korolev et al., 2002) therefore its proportions should be
optimized to prevent efficiency reduction of PCR caused by spermine
overdose. However, it is important to bear in mind that other inhibitory
compounds present in biological samples with affinity to spermine will
most likely alter the 1:4 ratio. Especially, this could be the case
whenever low concentrations of spermine are required. In order to
determine the optimal spermine concentration needed to be added to
samples with unknown DSS concentration a simple fluorometric
method was described herein. It was shown that both DSS and spermine
interfere with fluorometric measurement of DNA as used in e.g. the
Qubit technology. Addition of both DSS and spermine in optimal con-
centrations enables recovery of the fluorometric signal to the highest
level. Hence, DNA or cDNA samples contaminated with an unknown
concentration of DSS can be tested for the optimal amount of spermine
that is required in a given assay. By use of qPCR we show that DSS
inhibition of DNA polymerases could be completely counteracted by
addition of adequate amounts of spermine. Even though the excess of
spermine (above 0.01 g/L) in the qPCR reaction was demonstrated to
affect the PCR efficiency, Illumina amplicon sequencing revealed that
samples exposed to the same concentration of spermine were affected in
a highly similar manner. Qualitative analysis showed that all OTUs
could be recovered regardless the differences in spermine concentra-
tion. Weighted UniFrac analysis showed clustering of technical re-
plicates according to spermine concentration. This indicates dosage-
dependent changes in PCR efficiency that seemed to be recurrent for
given concentration of spermine. It is important to notice that differ-
ences in relative abundance although significant, were minor (Suppl.
Fig. 3). Similar or higher differences can often be seen between biolo-
gical or even technical replicates (Zhou et al., 2011).
In summary, we present here an easy and highly cost-effective
method to counteract inhibition of PCR and two step RT-qPCR caused
by DSS. To our knowledge this is the first reported method allowing for
successful counteracting DSS based inhibition of DNA polymerases.
Furthermore, we demonstrate here a simple approach to adjust the
concentration of spermine ensuring optimal DNA and cDNA based PCR
efficiency required during quantitative and semi-quantitative assays.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.mimet.2017.10.015.
Ethics approval and consent to participate
The study was carried out in agreement with Directive 2010/63/EU
of the European Parliament and of the Council of 22 September 2010 on
the protection of mice used for scientific purposes, and the Danish
6
Journal of Microbiological Methods 144 (2018) 1–7
Animal Experimentation Act (LBK 474 15/05/2014). The study was
approved by the Animal Experiments Inspectorate under the Ministry of
Environment and Food in Denmark (License: 2007-561-1434 C3).
Availability of data and material
The raw sequencing dataset has been submitted to European
Nucleotide Archive (ENA) under accession number # PRJEB17622.
Competing interests
The authors declare that they have no competing interests.
Funding
ŁK was partly funded by the Danish Strategic Research Council
(NEOMUNE, grant no 12-132401).
Authors' contributions
ŁK carried out the molecular tests, analysed the data and drafted the
manuscript. WK collected the sequencing data, participated in the
molecular assays and drafted the manuscript. KMB collected the murine
samples and participated in drafting of the manuscript. AKH partici-
pated in the study design and helped to draft the manuscript. FKV
conceived the original idea of using spermine to prevent inhibition of
PCR, coordinated the molecular trials and participated in the study
design. DSN coordinated the study and helped to draft the manuscript.
All authors read and approved the final manuscript.
Acknowledgments
We would like to express our sincere gratitude for Associate
Professor Henrik Siegumfeldt and Mr. Basheer Yousef Aideh for valu-
ables clues and technical assistance during our work.
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