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qPCR: avoiding signals in the no-template control

This months question from the Molecular Biology Forums (online at molecularbiology.
forums.biotechniques.com) comes from the Real-time qPCR/qRT-PCR Methods section.
Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or
the opinions of, BioTechniques.
How can I avoid technical replicate variation and signals in my no-template
controls? (Thread 21619)

Q.

I am having some problems with qPCR using plant RNA. I see amplification in the
no-template control (NTC) reactions and unacceptable technical replicate differences
in the Ct values.
I tried total RNA extraction and using Dynabeads from Invitrogen and prefer the latter
because I get better-quality RNA as determined by Nanodrop and Bioanalyzer. I reversetranscribe using Superscript II and oligo(dT)20. I am using the MX3000P system from
Stratagene and SYBR Green II. The genes form secondary structures and the genome is
duplicated. Because of this, I was very careful with the primer design and tried several primer
dilutions. Adding 2.5% DMSO helped very little and using more DMSO inhibited the
reaction. I checked every step on a gel and sequenced the PCR products. I verified that there
isnt genomic DNA contamination by PCR with suitable primers. I guess the variability is
coming from poor-quality cDNA. How can I solve this problem?

A The amplification you are seeing may actually be primer dimer and not real amplification.
Primer dimers still have melting peaks. If this is your problem, you might try reducing the
amount of primer you use.

If the problem is with the cDNA as you suspect, you could try using a mix of random primers
and oligo(dT). Qiagen supplies this option with the QuantiTect reverse transcription enzyme
and Bio-Rad offers it with their iScript enzyme.

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A Unacceptable replicates are usually caused by pipetting errors. ROX dye should normalize
for differences in the mastermix. I get very tight replicates using the Qiagen SYBR green
mastermix.

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Is your instrument calibrated and running properly?

Q Im using a ROX dye. It helps a little in correcting the signal, but the results arent affected
much by its presence. I see melting curves for the ROX channel, the NTC, and the NTC
SYBR Green signal.

I tried running a few plates on an Opticon machine but got similar results. The instruments are monitored by the technician at the core facility and other users havent reported
problems. I tried running the genes in different positions on the plate and still got variable
results, so it doesnt seem to be a problem with the machine.

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A The ROX channel cannot have a melting peak. The melting peak is caused by the change
in fluorescence as SYBR green binds dsDNA. As the temperature goes up and melts the
strands, the SYBR is released and stops fluorescing. ROX is not going to do that since it
doesnt bind DNA. If you see a peak in your water control, then you have contamination.
That could cause variability if different samples have varying levels of contamination.

After you make a batch of cDNA, do you dilute it for qPCR or do you dilute the RNA and
then perform the cDNA reactions? I recommend the first way.
Vol. 50 | No. 4 | 2011

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Q I dont dilute the RNA, but instead reverse-transcribe and then dilute the cDNA.
their experimental treatments affect cells
A
Your NTCstatus.
signal might only be from the primer-dimer
product,
so it may actually be
Eur. J. Biochem.
7:471-484.
bioenergetic

the cristae membrane of rat liver mitochondria.

negative. It is common to see dimers form when there

is no actual
template
for the
11. Nicholls,
D.G.
1974. The
infprimers
luence to
of
ATPishydrolysis
on the protonwork on. You may also see this in the high-dilutionrespiration
wells sinceand
there
little template.
Right
electrochemical
gradient
across the
after
annealing, you can add another data acquisition
step at a higher
temperature
thaninner
the
Acknowledgments
membrane of rat-liver mitochondria as deterextension
step.
That
should
melt
the
dimers
so
that
you
only
read
specific
signals.
The authors thank the reviewers for their
mined by ion distribution. Eur. J. Biochem.
helpful suggestions on this manuscript, and
50:305-315.
If
your contamination
is awhose
PCR product,
you can
usingkdUTP
With
12.try
Sz abad
a i, G.inathe
nd qPCR
M .R . mix.
Duchen.
apologize
to all colleagues
mitochondUTP,
the PCR
product
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strand. That allows
to perform
2008.
the hubyou
of cellular
Ca2+
drial work
we could
notwill
citehave
in this
review
signaling.
Physiology
(Bethesda)
23:84-94.
adue
2-min
digest
with
an
uracil-N-glycosylase
before
activating
the
Taq
DNA
polymerase.
Any
to space constraints. This work was
13. Szewczyk, A., W. Jarmuszkiewicz, and
contaminating
products will
be digested.
supported by PCR
the National
Institute
of
W.S. Kunz. 2009. Mitochondrial potassium
Health (NIH; grant nos. R21MH084718
channels. IUBMB Life 61:134-143.
andPhenolics
R21DA030256
to S.W.P.,
T32ES07026in your
14.original
Nic ho l plant
l s , D.G
. 2 0can
0 2 carry
. M iover
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onA
and possibly
polysaccharides
extracts
drial
function and dysfunction in the cell: its
to J.P.N.,
to E.B.B.,
your
RNA1DP2OD006501-01
samples and hurt the efficacy
of your RT
reactions.
relevance to aging and aging-related disease.
and 5R01MH056838 and 2P01MH64570
Int. J. Biochem. Cell Biol. 34:1372-1381.
to H.A.G.). This paper is subject to the
15.
Duchen,
M.R . 2that
0 0 4 .eliminated
M itochond
ria
Q
I
added
an
extra
fluorescence
reading
step
at
81C
after elongation
most
NIH Public Access Policy.
in health and disease: perspectives on a new
of the signal in the NTC amplifications.

mitochondrial biology. Mol. Aspects Med.

25:365-451.
I wonder if the slightly different melting temperatures
areiths,
caused
by secondary
structures
or
16. Griff
E.J.
20 0 0. Mitochond
ria-folding
the cDNA
that prevents
ofinmy
potential role
cellproducts.
life and death. Cardiovasc.
S.W.P., in
J.P.N.,
and H.A.G.
holdconsistent
patent amplification
Res. 46:24-27.
filings related to treatment of NeuroAIDS
17. Solaini, G., G. Sgarbi, G. Lenaz, and A.
using
of mitochondrial
hyper- of splice
Youinhibitors
might want
to explore the possibility
variants
your targets
of interest.
Baracca.
2007.ofEvaluating
mitochondrial
polarization.
Are
you sure of your target sequences?
membrane potential in cells. Biosci. Rep.
27:11-21.
18. Nichol ls, D.G. 2 0 0 6 . Si mu lta neous
Invitrogen has a reverse transcriptase called Thermoscript
can be used
higher
monitoring ofthat
ionophoreand at
inhibitormediated
plasma
and mitochondrial
temperatures
remove secondary
Qiagen
reverse
transcriptase
readsmembrane
through
1. Ehrenberg,to
B.,help
V. Montana,
M.D. Wei,structure.
J.P.
potential changes in cultured neurons. J. Biol.
secondary
Wuskell,structures.
and L.M. Loew. 1988. Membrane
Chem. 281:14864-14874.
potential can be determined in individual cells
19. Wa r d , M .W. , A . C . R e g o , B . G .
from the nernstian distribution of cationic
Frenguelli,
and D.G. Nicholls.
It is Biophys.
documented
in papers by S.A. Bustin (2002,
J. Mol. Endocrinol.
29:23-39)2000.
and
dyes.
J. 53:785-794.
Mitochondrial
membrane
and
2. Nicand
hol lMedrano
s , D.G . (2005,
a nd BioTechniques
M .W. Wa rd . 39:75-85)
Wong
that the biggest
sourcepotential
of variation
excitotoxicity
in cultured
cerebellar
2000. Mitochondrial
membrane
potential
in qPCR
is the person doing
the experiment.
Youglutamate
may be missing
something
simple
like
granule cells. J. Neurosci. 20:7208-7219.
and mixing
neuronal
glutamate
excitotoxicity:
proper
before
dispensing
into the reaction
to
20. tubes.
S c a d uPlates
t o , and
R . Ctubes
. , J are
r . very
a n dprone
L .W.
mortality and millivolts. Trends Neurosci.
cross-contaminations.
I
would
suggest
leaving
empty
wells
around
your
NTCs
and
vortexing
Grotyohann.
1999.
Measurement
of
mitochon23:166-174.
everything.
drial membrane potential using fluorescent
3. Lemasters, J.J. and V.K. Ramshesh. 2007.
rhodamine derivatives. Biophys. J. 76:469Imaging of mitochondrial polarization and
477.
depolarization with cationic fluorophores.
I agree with
the previous
comment: vortexing
21. reagents
S a l v i o l ibefore
, S . , useA is
. key.
A r dWhite-welled
izzoni, C.
Methods
Cell Biol.
80:283-295.
plates
reflectivity,
so Franceschi,
they give higher
signal, tighter
and A. Cossarizza.
1997.replicate
JC-1, but
4. Johfrom
n son,Eppendorf
L .V., Mhave
. L . higher
Wa l sh,
B .J.
not DiOC6(3)
123,
a reliable
Cts,Bockus,
and areand
cheaper
than1981.
otherMonitoring
plates. Theofonly drawback
is thatoritrhodamine
is harder to
seeisthe
wells
L.B. Chen.
fluorescent probe to assess delta psi changes
relative
in
where
youmitochondrial
have alreadymembrane
dispensedpotential
your reagents.
in intact cells: implications for studies on
living cells by fluorescence microscopy. J. Cell
mitochondrial functionality during apoptosis.
Biol. 88:526-535.
FEBS Lett.
411:77-82.
You might
autoclaving
your diluent
solution
again.
Reagent contamination is a
5. Perry,
S.W.,also
J.P. try
Norman,
A. Litzburg,
D.
22. Mathur,
Hong,water
B.K. or
Kemp,
Zhang,
Dewhurst,
and H.A.
chief
causeS.of
NTC signal
andGelbard.
usually 2005.
the suspect
reagent isA.,
theY.diluent
TE. A.A.
Barrientos, and J.D. Erusalimsky. 2000. EvaluHIV-1 transactivator of transcription protein
ation of fluorescent dyes for the detection of
induces mitochondrial hyperpolarization
mitochondrial
membrane
and synaptic
stress
apoptosis.
You
might want
to leading
put yourtosamples
intoJ.maximum
recovery tubes
or usepotential
a carrier changes
nucleic
in cultured
cardiomyocytes. Cardiovasc. Res.
Immunol.
acid.
Or you174:4333-4344.
could try another type of detection like
TaqMan.
46:126-138.
6. Rottenberg, H. and S. Wu. 1998. Quantitative
23. Chinopoulos, C., L. Tretter, and V.
assay by flow cytometry of the mitochondrial
1999.
Depolarization
of in the
situ
membrane
in intact
cells.signals
Biochim.
You
mightpotential
avoid primer
dimer
by settingAdam-Vizi.
the detection
temperature
between
due to hydrogen peroxideBiophys.
Acta 1404:393-404.
melting
temperature
of the dimer and the melting mitochondria
temperature
of
the
target.
induced oxidative stress in nerve terminals:
7. Nicholls, D.G. and S.L. Budd. 2000.
inhibition of alpha-ketoglutarate dehydroMitochondria and neuronal survival. Physiol.
TheRev.
most
likely causes of your variable results in the replicates
are that the73:220-228.
plate is not properly
genase. J. Neurochem.
80:315-360.
sealed,
your target gene
is low (Ct 3540,
dynamic range
Mx3000P
or similar
24. Scanlon,
J.M. with
and I.J.
Reynolds.
1998.
8. Abdel-Hamid,
K.M.expression
and M. Tymianski.
and is
glutamate
instruments
is poorerand
thaneffects
with ABI7900),
qualityofofoxidants
your RNA
not good.receptor
1997. Mechanisms
of intracel- or the Effects
activation on mitochondrial membrane
lular calcium buffering on neuronal survival
potential in rat forebrain neurons. J. Neurochem.
in
organotypic
hippocampal
cultures
exposed
Selected and edited by Kristie Nybo, Ph.D.
71:2392-2400.
to anoxia/aglycemia or to excitotoxins. J.
25. Buck ma n, J.F. a nd I.J. Rey nolds.
Neurosci. 17:3538-3553.
BioTechniques
50:213-215
(April
2001. Spontaneous changes in mitochondrial
9. Reid, R.A.,
J. Moyle,
and2011)
P. Mitchell. 1966.
membrane potential in cultured neurons. J.
doi 10.2144/000113648
Synthesis of adenosine triphosphate by a
Neurosci. 21:5054-5065.
protonmotive force in rat liver mitochondria.
26. Moudy, A.M., S.D. Handran, M.P.
Naturereprints
212:257-258.
To purchase
of this article, contact: biotechniques@fosterprinting.com
Goldberg, N. Ruffin, I. Karl, P. Kranz-Eble,
10. Mitchell, P. and J. Moyle. 1969. Estimation of
D.C. DeVivo, and S.M. Rothman. 1995.
membrane potential and pH difference across

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