Analytical Biochemistry 400 (2010) 301–303

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Analytical Biochemistry
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Notes & Tips

Mutant of Moloney murine leukemia virus reverse transcriptase exhibits

higher resistance to common RT-qPCR inhibitors
Bahram Arezi *, Melissa McCarthy, Holly Hogrefe
Agilent Technologies, Stratagene Products Division, La Jolla, CA 92037, USA

a r t i c l e i n f o a b s t r a c t

Article history: Inhibitor resistance of several commercial Moloney murine leukemia virus reverse transcriptase (MMLV
Received 15 December 2009 RT) enzymes was investigated. IC50 values were determined for potential RNA contaminants, including
Received in revised form 5 January 2010 guanidine thiocyanate, ethanol, formamide, ethylenediaminetetraacetic acid (EDTA), and plant-related
Accepted 19 January 2010
acidic polysaccharides. Sensitivity (as judged by MMLV RT IC50 values) was directly correlated to the out-
Available online 25 January 2010
come of ‘‘mock” reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays carried
out with exogenous inhibitors. MMLV RT enzymes lacking RNase H activity were shown to be more sen-
sitive to RT-qPCR inhibitors. In contrast, a thermal-resistant MMLV RT pentuple mutant (E69K/E302R/
W313F/L435G/N454K) showed higher tolerance to these substances than the wild type. Increased resis-
tance was also noted in RT-qPCR comparisons employing crude cell lysates.
Ó 2010 Elsevier Inc. All rights reserved.

Reverse transcription is the first step in reverse transcription-
quantitative polymerase chain reaction (RT-qPCR);1 thus, any
impairment in reverse transcriptase (RT) activity due to the presence
of inhibitors can lead to significant error in quantification or produce
false-negative data. Potential RT inhibitors include chemicals used in
RNA isolation, such as guanidine and ethanol, and endogenous sub-
stances that copurify during RNA extraction, such as plant-related
polysaccharides. Sensitivity of commercial RT enzymes to inhibitors
is becoming increasingly important as researchers strive to use smal-
ler and less pure sources of RNA, such as micro-dissected or forma-
lin-fixed paraffin-embedded tissue and cell extracts prepared with
commercial lysis reagents.
Exogenous RNA controls are commonly used in RT-qPCR exper-
iments to normalize variation caused by the presence of copurified
inhibitors [1–3]. When using such controls, however, one assumes
that amplifications of target and control sequences are inhibited to
the same extent, which is not always the case [4]. When the pres-
ence of inhibitors is suspected, researchers can repurify or dilute
the RNA sample so long as the RNA amount and/or target abun-
dance are not limiting. An easier and more reliable strategy is to
* Corresponding author. Fax: +858 373 5300.
E-mail address: bahram.arezi@agilent.com (B. Arezi).
1
Abbreviations used: RT-qPCR, reverse transcription-quantitative polymerase chain
reaction; RT, reverse transcriptase; MMLV, Moloney murine leukemia virus; DMSO,
dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecyl
sulfate; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; cDNA, com-
plementary DNA; GAPD, glyceraldehyde-3-phosphate dehydrogenase; AMV, avian
myeloblastosis virus.
use RT enzymes that are more broadly tolerant of RNA contami-
nants. A previous study reported that a commonly used RNase H-
minus Moloney murine leukemia virus (MMLV) RT mutant (Super-
Script II) loses complete activity in the presence of formamide
(15%, v/v), dimethyl sulfoxide (DMSO, 25%, v/v), guanidine isothio-
cyanate (100 mM), ethylenediaminetetraacetic acid (EDTA,
2.5 mM), and sodium dodecyl sulfate (SDS, 0.005%, w/v) [5].
In this article, we compare an expanded group of MMLV RT en-
zymes with respect to inhibitor resistance and RT-qPCR perfor-
mance with crude RNA preparations. The enzymes tested include
wild-type MMLV RT and three commercial MMLV RT mutants with
increased thermal activity: Mut1 (AffinityScript RT from Agilent
Technologies/Stratagene Products, MMLV RT E69K/E302R/W313F/
L435G/N454K [6]), Mut2 (SuperScript II RT from Life Technologies,
RNase H-minus RT), and Mut3 (SuperScript III RT from Life Tech-
nologies, thermostable RNase H-minus RT). Mutations that elimi-
nate RNase H activity also provide increased thermal resistance
compared with wild-type MMLV RT, and this is attributed to great-
er stabilization provided by intact (associated with Mut2 and
Mut3) versus degraded (associated with wild type and Mut1)
RNA [7]. Like Mut1, Mut3 was engineered for increased activity
at higher temperatures; however, the mutations employed and
the basis for increased thermal resistance are distinctly different.
In addition to RNase H-minus mutations, Mut3 contains multiple
mutations in the polymerase domain that increase thermal resis-
tance independent of template binding [8]. In contrast, Mut1 is
protected from heat denaturation through tighter binding to tem-
plate–primer (Km [RNA–DNA]: 1 lM, MMLV RT; 0.1 lM, Mut1 [ki-
netic measurements obtained in the absence of RNase H activity])
[6]. Compared with wild-type enzyme (t1/2 < 5 min), the half-lives

0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.01.024
302 Notes & Tips / Anal. Biochem. 400 (2010) 301–303
of MMLV RT mutants range from 10 min (Mut2) to more than
20 min (Mut1 and Mut3) when measured at 55 °C in the presence
of template–primer (data not shown).
To quantify inhibitor resistance, IC50 values were determined for
a number of potential RNA contaminants, including guanidine thio-
cyanate (Fluka, Cat. No. 50983), ethanol (Pharmco–AAPER), formam-
ide (Mallinckrodt Baker, Cat. No. 3797), EDTA (Spectrum, Cat. No.
E1040), pectin (Sigma, Cat. No. 76282), and xylan (Sigma, Cat. No.
X4252). Reactions (in duplicate) contained 200 lM dATP, dGTP,
and dCTP, 100 lM TTP, 2 lCi 3H-labeled TTP, 15 pmol of primer
(50 -ACACCCAGTTGTAGTAGGACACC-30 ), 1.5 pmol of RNA template
(500 nt in vitro transcribed RNA, Agilent Technologies/Stratagene
Products, Cat. No. 252561), and 0.5 pmol of RT in RT buffer consisting
of 50 mM Tris–HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2. Dithio-
threitol (DTT) was added to a final concentration of 5 mM (Mut3)
or 10 mM (wild type, Mut1, and Mut2). Inhibitors were added in a
range of concentrations bracketing predicted IC50 values. Reactions
were incubated for 5 min at the manufacturer’s recommended tem-
perature of 37 °C (MMLV RT), 42 °C (Mut1 and Mut2), or 50 °C
(Mut3) and then were stopped by adding EDTA (45 mM, final). IC50
values were determined using equimolar amounts of RT (0.5 pmol
as judged by equal staining intensity on SDS–polyacrylamide gel
electrophoresis [PAGE] gels) to control for differences in unit deter-
minations between different enzyme manufacturers (0.5 pmol cor-
responds to 10 U of MMLV RT, Mut1, and Mut3 and to 14 U of
Mut2 based on label unit concentrations).
The impact of RT inhibitors in RT-qPCR was evaluated with
complementary DNA (cDNA) generated in the presence of various
chemicals (‘‘spike-in” experiments) or synthesized from crude cell
extracts. cDNA synthesis reactions (20 ll) contained 500 ng of oli-
go(dT)18, 4 mM dNTPs, 100 ng of qPCR Universal Reference RNA
(Agilent Technologies/Stratagene Products, Cat. No. 750500), and
either 50 or 200 U of RT in RT buffer. Formamide was added to
cDNA synthesis reactions (3–12%, v/v), and samples were incu-
bated for 60 min at the recommended temperature. A portion
(2 ll) of each cDNA sample was amplified with Brilliant II qPCR
Master Mix (Agilent Technologies/Stratagene Products, Cat. No.
600804) using the Mx3005P Real-Time PCR System (Agilent Tech-
nologies/Stratagene Products, Cat. No. 401449) and 200 nM
ADAM 17 primers/probe (forward primer: 50 -GAAGTGCCAGG
AGGCGATTA-30 ; reverse primer: 50 -CGGGCACTCACTGCTATTACC-
30 ; probe: FAM-TGCTACTTGCAAAGGCGTGTCCTACTGC-BHQ1). Cell
extracts were prepared by lysing Jurkat cells with SideStep Lysis
and Stabilization buffer (Agilent Technologies/Stratagene Prod-
ucts, Cat. No. 400900) according to the manufacturer’s protocol.
Samples containing 50–2500 cell-equivalents in 0.5 ll of lysis
buffer were added to cDNA synthesis reactions consisting of
500 ng of oligo(dT)18, 4 mM dNTPs, and 50 U of RT in RT buffer.
RT-qPCR was carried out as described above using 1 glyceralde-
hyde-3-phosphate dehydrogenase (GAPD) primers/probe (Life
Technologies Assays-on-Demand high-abundance target).
A comparison of IC50 values (Table 1) reveals that Mut1 toler-
ates higher concentrations of guanidine thiocyanate (2.6-fold),
Table 1
IC50a values for suspected reverse transcriptase inhibitors
RT Guanidine (mM) Formamide (% v/v)
MMLV 42.9 8.7 ± 0.4
MMLV(bMut1) 111.3 ± 8.6 15.3 ± 0.7
MMLV(cMut2) 29.5 7.3 ± 0.1
MMLV(dMut3) 28.7 7±2
a
IC50 values were determined from plots of reverse transcriptase activity (nucleotide incorporation) versus inhibitor concentration. When Rsq of the linear regression plot
was less than 0.95, the experiment was repeated one or two more times, and the corresponding standard deviations were reported.
b
Mut1 is AffinityScript RT, an MMLV RT with mutations E69K, E302R, W313F, L435G, and N454K.
c
Mut2 is SuperScript II RT, an RNase H-minus MMLV RT with three mutations in the RNase H domain (8).
d
Mut3 is SuperScript III RT, an RNase H-minus MMLV RT with three mutations in the RNase H domain and four mutations in the polymerase domain (8).
formamide (1.8-fold), ethanol (2.4-fold), xylan (3.6-fold), and pec-
tin (3.9-fold) compared with wild-type MMLV RT. In contrast,
Mut2 and Mut3 appear to be more sensitive than wild-type MMLV
RT to inhibition by organic solvents (1.2- to 1.4-fold) and acidic
polysaccharides (5- to 7-fold). Mechanisms of inhibition are largely
speculative, but relative activity in organic solvents likely reflects
intrinsic stability (resistance to protein denaturants), whereas sen-
sitivity to acidic polysaccharides may indicate competitive inhibi-
tion of template–primer binding to the RT active site. As
discussed earlier, tighter binding to template–primer protects
Mut1 against thermal denaturation and likely explains reduced
sensitivity to protein denaturants and competitive substrate inhib-
itors in nucleotide incorporation assays. In contrast, Mut2 and
Mut3 bind template–primer with similar affinity to wild type
[7,9], and in this case one might expect inhibitor resistance to par-
allel thermal resistance in the absence or presence of RNA. As
shown in Table 1, however, Mut2 and Mut3 are equally sensitive
to inhibitors despite different temperature optima and are slightly
(protein denaturants) or substantially (acidic polysaccharides)
more sensitive to inhibitors compared with wild-type MMLV RT.
These results suggest that RNase H proficiency may play a role in
inhibitor resistance (e.g., conformational changes accompanying
RNA degradation or presence of template–primer in the RNase H
domain may reduce nonspecific binding of acidic polysaccharides)
or that one of the RNase H-minus mutations reduces selectivity for
template–primer.
The impact of inhibitors on reverse transcription was also inves-
tigated by qPCR. cDNA synthesis was performed with wild-type
and mutant MMLV RT enzymes in the presence of varying concen-
trations of formamide, xylan, guanidine thiocyanate, and ethanol
(Fig. 1A and data not shown). qPCRs employed identical conditions
and reagents (Brilliant II qPCR Master Mix) to ensure that Cq (quan-
tification cycle) differences are directly related to inhibitor sensi-
tivity of MMLV RT enzymes. We used higher RT concentrations
in these experiments (50 U of RT, equivalent to 2.5 pmol of MMLV,
Mut1, and Mut3 and to 1.8 pmol of Mut2) to mimic the amount of
RT that is usually used in RT-qPCR. Results obtained with 50 U of
RT showed the following sensitivity to inhibition by formamide
(from lowest to highest): Mut1 > Mut2 P MMLV P Mut3
(Fig. 1A), which paralleled relative differences in IC50 values for
formamide (>15%, v/v): Mut1 > MMLV P Mut2 P Mut3 (7%, v/v).
Not surprisingly, inhibition with formamide was overcome to
some extent by using 200 U of Mut2 and Mut3 (DCq between 0%
and 9% formamide: 50 U of Mut2, 8.1; 200 U of Mut2, 4.1; 50 U
of Mut3, 9.3; 200 U of Mut3, 4.6). However, higher RT amounts
(200 vs. 50 U) can be inhibitory to RT-qPCR, as shown here by Cq
delays of 2.1 (Mut2) and 3.9 (Mut3) in the absence of formamide.
Moreover, PCR inhibition at high RT concentrations is well docu-
mented in the literature [10,11]. When xylan, guanidine thiocya-
nate, and ethanol were spiked into reverse transcription
reactions, we also observed a direct correlation between inhibitor
sensitivity (as judged by MMLV RT IC50 values) and Cq delays with
increasing concentrations of denaturant (data not shown).
Ethanol (% v/v) EDTA (mM) Xylan (% w/v) Pectin (% w/v)
13.1 ± 1 2.6 ± 0.3 0.035 0.27
31.2 ± 6.4 2.8 ± 0.1 0.127 1.05
10.5 ± 0.9 2.05 ± 0.1 0.005 ± 7e5 0.071
10.7 ± 0.1 1.7 ± 0.05 0.0075 0.094
 Notes & Tips / Anal. Biochem. 400 (2010) 301–303 303

A 4 4
M
Mut11
0.753; Mut2, 0.683; Mut3, 0.754), and inflated amplification effi-
Fluuorescence MMMLVV
(dRRn) (550U
U/Rxxn) ciencies (wild type, 385.7%; Mut2, 340.5%; Mut3, 352.6%) result-
(50U
U/R
Rxn))
112%
% ing from compression of Cq values at more than 250 cell-
2 99% 2 %
12% equivalents. Amplifications of additional lower abundance targets
6%
0, 33% 00, 3, 6, 99% (ABCC, ADAM 17) produced similar results (data not shown), indi-
cating that Mut1 is more resistant than other MMLV RT enzymes
110 20 330 40 10 200 30 400 to inhibitors in lysed cells and, therefore, can be used in combina-
C
Cyclees Cycles
4 4 tion with crude RNA samples (e.g., commercial cell-to-RNA re-
Muut2 M
Mut2
Fluuorescence
(50U
U/RRxn)) (2200U
U/Rxxn)
agents) to generate high-quality quantitative data across a
(dRRn) 122%
12% broader range of sample sizes.
9%%
2
6%
2
99%
In summary, we have demonstrated that a pentuple mutant of
MMLV RT (Mut1) exhibiting higher affinity for template–primer
00, 3% 0, 33, 6%
%
and increased thermal activity also provides increased tolerance
110 20 330 40 10 200 30 400
to common RT-qPCR inhibitors. As shown here, inhibitor resistance
C
Cyclees Cycles is an important attribute of RTs used with small or less pure
4
Muut3
4
M
Mut3
sources of RNA that allows researchers to eliminate RNA purifica-
Fluuoresscencce
(dRRn) (50U
U/RRxn)) (2200U
U/Rxxn) 122% tion (or repurification) and dilution steps from RT-qPCR proce-
%
12%
9%
% %
9% dures. In the future, we anticipate that inhibitor-resistant RTs
2 2
%
33-6% 66% will be used in combination with PCR enzymes, similarly engi-
3% neered for increased tolerance to polymerase inhibitors [12,13],
00% 0%
%
to amplify crude RNA samples with high efficiency. In light of
110 20 330 40 10 220 30 440 our findings, it would be of interest to investigate inhibitor resis-
C
Cyclees Cyccles
tance of avian myeloblastosis virus (AMV) RT, which (like Mut1)
B exhibits higher thermal activity and template–primer affinity com-
33 pared with MMLV RT [7].

Cq
31
(dRn)
29
27
25
100
Initial quantity (cell-equivalent number)
Fig. 1. Effect of RT inhibitors in real-time PCR. Inhibitors were added to cDNA
synthesis reactions containing the indicated MMLV RT enzymes, and qPCR was
performed as described in the text. (A) Formamide was added in amounts ranging
from 3% to 12% (v/v). Amplification plots of ADAM 17 target show dRn values
consisting of baseline-subtracted and normalized (to ROX passive reference)
fluorescence signal. (B) Jurkat cells were lysed with SideStep buffer (no inhibition
of RT-qPCR with up to 5 ll of buffer) and cDNA was prepared as described from 50
to 2500 cell-equivalents. GAPD standard curves are shown along with correspond-
ing R2 values and regression fit equations.
Finally, inhibitor resistance of MMLV RT enzymes was com-
pared in qPCR reactions employing reverse-transcribed crude cell
lysate. Jurkat cells were treated with SideStep buffer, which effec-
tively lyses tissue culture cells and stabilizes RNA, eliminating the
need for RNA purification. As shown in Fig. 1B, standard curves
(GAPD target) generated from Mut1-transcribed samples are lin-
ear across the range of 50–2500 cell-equivalents (R2 = 0.951,
amplification efficiency = 116.6%). In contrast, lysates that were
reverse transcribed with wild-type, Mut2, and Mut3 MMLV RTs
produced standard curves with poor linearity due to inhibition
at more than 250 cell-equivalents, as shown by Cq delays (e.g.,
3 cycles at 2500 cell-equivalents), lower R2 values (wild type,
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