TIBTEC 1738 No. of Pages 14
TIBTEC 1738 No. of Pages 14

Validating a Quantitative PCR (qPCR) Experimentto Minimize Error and Maximize Data QualityThesolu�on:The
solu�on:The
problem:The
problem:
(A)
(B)
(B)
(A)
Primervalida�on
to todetermine
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Table
1.
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ApproachtoPerforming
a
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qPCR
ExperimentThe
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valuesThe
problem:
(A)
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of of
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acids
in in
each
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Description
stepsof nucleic acids in each sample

Step
1

Experimental 1 1 Procedure
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Nonvalidated
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atcorrect
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Nonvalidated,unstablereference
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appropriate dilu�on factor (see step #1B)
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standard
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Low
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least
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110%)
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eachtargetforfortheexperiment(i.e.,
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poorlyvalidatedreference
genes.
group).
TrendsinThisBiotechnology,
Month
Year,
Vol. xx, No.
yycontaminants
https://doi.org/10.1016/j.tibtech.2018.12.002
that the pooled DNA sample represents the average abundance of each target for the experiment (i.e., equalized pool of the same number of DNA samples from each biological©qPCR
2018data
The
Authors.
Publishedby Elsevier when
Ltd.
This
is
an open accessarticleunderusing
theCCa BY-NC-ND
licenseunstable(http://creativecommons.org/licenses/by-nc-nd/4.0/).
canchangedramaticallynormalization
is isperformed
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group).
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from [8].
qPCR data can change dramatically when normalization is performed using a stable versus unstable reference gene.

Figure 4. Validating a Quantitative PCR (qPCR) Experimentto Minimize
and3A).
Maximize Data Quality. (A) Nonvalidated primers could give variable and/or
controlsError(Figurewith required

Trendsin in
Biotechnology,
Month
Year,
Vol.
xx,xx,
No.
yyyy 7 7
TrendsBiotechnology,
Month
Year,
Vol.
No.

TIBTEC 1738 No. of Pages 14

Stepwise Approach to Performing a Valid qPCR Experiment a
Table 1. Stepwise Approach to Performing a Valid qPCR Experimenta
Step

Description

Sub steps

Instruction

1

Experimental

Procedure

List the best targets, samples, and treatments based on previously acquired, vetted, and validated data or

Step

Description

Sub steps
Biologicalgroups1

Experimental
designProcedure

Instruction knockout, time points, etc.) and various
Deﬁne the appropriate biological groups (i.e., treatments,
thereof(Figure1).
combinations
List the best targets, samples, and treatments based on previously acquired, vetted, and validated data or
1A). number of animals or number of cell culture plates per biological group, which isliterature
Biological(Figure(samples):a
Table 1. Stepwise
qPCR (Figure
Experiment
1A).
designApproach to Performing a Validliterature

Replicates

Biological groups
Replicates
Experimental
conditions
2

Tissue and cell
harvesting

Sample extraction
Experimental(complex organisms)
conditions
Sample extraction(cells) extraction
Samplewith the aidbiologicalof a biostatistician
and/ora statisticalpowertime
analysis(highly
determinedDeﬁnethe appropriategroups (i.e.,treatments,
knockout,
points,etc.)dependentand variouson thethe organism).
number of wells pipetted per cDNA sample from each biologicalcomplexity of thereof(Figure Technical:
1).
combinations
replicate (typically two or three); (Figure 1).
Biological (samples): number of animals or number of cell culture plates per biological group, which is
Carefully note
all thecontrollable
such as:lot consistency
cell culture
media,FBS,
BSA, animal
feed
with
aid of a factors,
biostatistician
and/ora statisticalinpower
analysis(highlydependent
on thedeterminedsex,
and phenotype.
picturesthroughout
the experimentandcarefully
note
and drugs orofcompounds,
the organism).
Technical:
numberTakeof wells
pipetted
per cDNA
sample fromeach
biologicalcomplexity
changessamples1).or specimens that may become outliers.
any unusual(typically
two inorspeciﬁcthree); (Figure
replicate
Sacriﬁce note
animals,
extract andfactors,
dissectsuch
tissueandinsequentially.
Flash freeze
in liquid
nitrogenCarefully
all controllable
as:reproducibly
lot consistency
cell culture media,
FBS, BSA,
animalfeed
dissection
1C).
immediately
drugs or upon
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an ODEnsure
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Test RNA
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withand may also be a useful normalization target [47].
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genes
and may also be a useful normalization target [47].

aTestfrom
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reference gene primers against three cDNA samples from each biological/treatment
Adapted and updated under aConﬁrm
Creativetarget
Commons license[8].
stability
group. Test stability using GeNorm, NormFinder and BestKeeper software. Normalize to multiple reference
genes (Figure 4D).
Trends in Biotechnology, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.tibtech.2018.12.002© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

10Trendsin Biotechnology,
Year, Vol.
xx, No. yylicense from [8].
Adaptedandupdated underMontha Creative
Commonsa