Nucleic Acids Res. 2002 May 1; 30(9): e36.

PMCID: PMC113859
Copyright 2002 OxIord University Press



Relative expression software tool (REST)
for group-wise comparison and statistical
analysis of relative expression results in
real-time PCR







Michael W. PIaIIl,
a
Graham W. Horgan,
1
and Leo DempIle
2

Institute oI Physiology, FML-Weihenstephan, Center oI LiIe and Food Sciences, Technical University oI
Munich, Germany,
1
Biomathematics and Statistics Scotland, Rowett Research Institute, Bucksburn, Aberdeen
AB21 9SB, UK and
2
Department oI Animal Science, Center oI LiIe and Food Sciences, Technical University
oI Munich, Germany
a
To whom correspondence should be addressed at present address: Institute oI Physiology, FML-
Weihenstephan, LiIe Science Zentrum Weihenstephan, Technische Universitaet Muenchen, Weihenstephaner
Berg 3, D-85350 FreisingWeihenstephan, Germany. Tel: 49 8161 71 3511; Fax: 49 8161 71 4204; Email:
pIaIIlwzw.tum.de
Received January 21, 2002; Revised March 4, 2002; Accepted March 4, 2002.



STRCT

Real-time reverse transcription Iollowed by polymerase chain reaction (RTPCR) is the most suitable method
Ior the detection and quantiIication oI mRNA. It oIIers high sensitivity, good reproducibility and a wide
quantiIication range. Today, relative expression is increasingly used, where the expression oI a target gene is
standardised by a non-regulated reIerence gene. Several mathematical algorithms have been developed to
compute an expression ratio, based on real-time PCR eIIiciency and the crossing point deviation oI an
unknown sample versus a control. But all published equations and available models Ior the calculation oI
relative expression ratio allow only Ior the determination oI a single transcription diIIerence between one
control and one sample. ThereIore a new soItware tool was established, named REST (relative expression
soItware tool), which compares two groups, with up to 16 data points in a sample and 16 in a control group,
Ior reIerence and up to Iour target genes. The mathematical model used is based on the PCR eIIiciencies and
the mean crossing point deviation between the sample and control group. Subsequently, the expression ratio
results oI the Iour investigated transcripts are tested Ior signiIicance by a randomisation test. Herein,
development and application oI REST is explained and the useIulness oI relative expression in real-time
PCR using REST is discussed. The latest soItware version oI REST and examples Ior the correct use can
be downloaded at http://www.wzw.tum.de/gene-quantiIication/.

TRODUCTO

Reverse transcription (RT) Iollowed by polymerase chain reaction (PCR) is a powerIul tool Ior the detection
and quantiIication oI mRNA. Nowadays real-time RTPCR is widely and increasingly used, because oI its
high sensitivity, good reproducibility and wide quantiIication range (1,2). It is the most sensitive method Ior
the detection and quantiIication oI gene expression levels, in particular Ior low abundance mRNA (1,2), in
tissues with low concentrations oI mRNA (e.g. bone marrow, Iatty tissues), Irom limited tissue samples (e.g.
biopsies, single cells) (3,4) and to elucidate small changes in mRNA expression levels (1,2,5). However, it is a
very complex technique with various substantial problems associated with its true sensitivity, reproducibility
and speciIicity and, as a Iully quantitative methodology, it suIIers Irom the problems inherent in real-time
RTPCR. Generally, two quantiIication strategies can be perIormed: an absolute and a relative quantiIication.
In absolute quantiIication the absolute mRNA copy number per vial or capillary is determined by comparison
with appropriate external calibration curves (2). An absolute quantiIication makes it easier to compare
expression data between diIIerent days and laboratories, because the calibration curve is a non-changing solid
and reliable basis. The relative expression is based on the expression ratio oI a target gene versus a reIerence
gene and is adequate Ior most purposes to investigate physiological changes in gene expression levels. Trends
can be better explained by relative quantiIication, but the results are strongly dependent on the reIerence gene
and the normalisation procedure used. Some mathematical models have already been developed to calculate
the relative expression ratios oI single samples (68;
http://docs.appliedbiosystems.com/pebiodocs/04303859.pdI), with or without eIIiciency correction. Equation
shows the most convenient mathematical model, which includes an eIIiciency correction Ior real-time PCR
eIIiciency oI the individual transcripts (6).

Ratio (
target
)
ACP
target(control sample)
/(
reI
)
ACP
reI(control sample)


The relative expression ratio oI a target gene is computed, based on its real-time PCR eIIiciencies () and the
crossing point (CP) diIIerence (A) oI an unknown sample versus a control (ACP
control sample
). In mathematical
models the target gene expression is normalised by a non-regulated reIerence gene expression, e.g. derived
Irom housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), albumin, actins, tubulins,
cyclophilin, 18S ribosomal RNA (rRNA) or 28S rRNA (911). But all published equations and available
models Ior the calculation oI relative expression ratios allow Ior the determination oI only a single
transcription diIIerence between one control and one sample (3 1), e.g. given in an DNA array experiment,
and not Ior a group-wise comparison Ior more samples (3 ~ 2), given in an experimental trial.

ThereIore, a new soItware tool was established, named REST (relative expression soItware tool), which
compares two groups, with up to 16 data points in the sample group versus 16 data points in the control group,
and tests the group diIIerences Ior signiIicance with a newly developed randomisation test. Nevertheless, the
successIul application oI real-time RTPCR and REST depends on a clear understanding oI the practical
problems. ThereIore, a clear experimental design, application and validation oI the applied real-time RTPCR
remains essential Ior accurate and Iully quantitative measurement oI mRNA transcripts. This paper explains
the development oI REST application, discusses the technical aspects involved in an experimental trial and
illustrates the useIulness oI relative expression in real-time RTPCR using REST.

TERS D ETHODS

Animal experiment, total RNA extraction and reverse transcription
Total RNA extraction was perIormed Irom rat liver as described previously (12). Adult rats were either Ied
with physiological zinc concentrations (control group, 58 p.p.m. Zn, 3 7) or suIIered 2229 days under zinc
depletion (sample group, 2 p.p.m. Zn, 3 6) (W.Windisch, manuscript submitted). Isolated total RNA
integrity was electrophoretically veriIied by ethidium bromide staining and by an average optical density
(OD) OD
260
/OD
280
nm absorption ratio oI 1.97 (range 1.782.09). Either 330, 1000 or 3000 ng total RNA was
reverse transcribed with 100 U Superscript II Plus RNase H

reverse trancriptase (Gibco LiIe Technologies,

Gaithersburg, MD) in a volume oI 40 l, using 100 M random hexamer primers (Pharmacia Biotech,
Uppsala, Sweden) according to the manuIacturers` instructions. ThereIore, concentrations oI 8.25, 25 or 75 ng
cDNA ( reverse transcribed total RNA) per l were achieved.

Optimisation oI RTPCR
Highly puriIied salt-Iree primer Ior target gene metallothionein (MT) (Iorward primer, CTC CTG CAA GAA
GAG CTG CT; reverse primer, TCA GGC GCA GCA GCT GCA CTT) and Ior reIerence gene GAPDH
(Iorward primer, GTC TTC ACT ACC ATG GAG AAG G; reverse primer, TCA TGG ATG ACC TTG GCC
AG) were generated commercially (MWG Biotech, Ebersberg, Germany). The MT primer set is able to
ampliIy the transcripts oI MT isoIorm 1 and MT isoIorm 2 mRNA. Conditions Ior real-time PCRs were
optimised in a gradient cycler (Mastercycler Gradient, EppendorI, Germany) with regard to %,6 DNA
polymerase (Roche Molecular Biochemicals, Basel, Switzerland), Iorward and reverse primers, MgCl
2

concentrations (Roche Molecular Biochemicals) and various annealing temperatures (5466C). RTPCR
ampliIication products were separated on a 4 high resolution NuSieve agarose (FMC Bio Products,
Rockland, ME) gel electrophoresis and analysed with the Image Master system (Pharmacia Biotech).
Optimised conditions were transIerred to the Iollowing LightCycler real-time PCR protocol.

LightCycler real-time PCR
For determination oI test and soItware variations all applications oI diIIerent total cDNA input were
perIormed in triplets (MT 13 and GAPDH 13). Real-time PCR mastermix was prepared as Iollows (to the
indicated end-concentration): 6.4 l water, 1.2 l MgCl
2
(4 mM), 0.2 l Iorward primer (0.4 M), 0.2 l
reverse primer (0.4 M) and 1 l LightCyclerFast Start DNA Master SYBR Green I (Roche Molecular
Biochemicals). Nine microlitres oI master-mix was Iilled in the glass capillaries and a 1 l volume oI cDNA
(either 8.25, 25 or 75 ng) was added as PCR template. Capillaries were closed, centriIuged and placed into a
cycling rotor. A Iour-step experimental run protocol was used: (i) denaturation program (10 min at 95C); (ii)
ampliIication and quantiIication program repeated 40 times (15 s at 95C; 10 s at 60C Ior MT or 10 s at 58C
Ior GAPDH; 20 s at 72C; 5 s at 86C Ior MT or 5 s at 84C Ior GAPDH with a single Iluorescence
measurement); (iii) melting curve program (6099C with a heating rate oI 0.1C per s and a continuous
Iluorescence measurement); (iv) cooling program down to 40C. To improve SYBR Green I quantiIication a
high temperature Iluorescence measurement point at the end oI the Iourth segment was perIormed (13). It
melts the unspeciIic PCR products below the chosen temperature, e.g. primer dimers, eliminates the non-
speciIic Iluorescence signal and ensures accurate quantiIication oI the desired GAPDH and MT real-time RT
PCR product, respectively. For the described mathematical model it is necessary to determine the CPs Ior
each transcript. The CP is deIined as the point at which the Iluorescence rises appreciably above the
background Iluorescence. In this study the Second Derivate Maximum Method was perIormed Ior CP
determination, using LightCycler SoItware 3.5 (Roche Molecular Biochemicals).

Statistics
For statistical evaluations oI the determined CP variations and calculated relative expression variations
(Tables (Tables113), data were analysed Ior signiIicant diIIerences by ANOVA using approximate tests
(Sigma Stat Ior Windows SoItware, Version 2.0; Jandel Corporation).



Table
ANOVA oI CV oI inter-assay variation oI MT and GAPDH CPs determined in rat liver by real-time RTPCR
started either with 8.25 (13), 25 (46) or 75 ng (79) cDNA per capillary



8aw C daLa seLs are ldenLlcal Lo 1able 1 AnCvA (o 9) was performed for M1 and CAuP separaLely Lo LesL Lhe
lnfluence of Lhe cunA sLarLlng concenLraLlon on expresslon raLlo wlLhouL normallsaLlon
Table 3
Factor oI down-regulation oI MT and GAPDH expression levels in rat liver under zinc depletion ( NOT
normalised by the GAPDH expression)

DLVLLCMLN1 CI kLS1

Cur goal was Lhe developmenL of a sofLware Lool LhaL allows for a relaLlve quanLlflcaLlon beLween groups and a
subsequenL LesL for slgnlflcance of Lhe derlved resulLs wlLh a sulLable sLaLlsLlcal model lurLher Lhe sofLware musL be
able Lo run on a wldely avallable plaLform whlch can be used worldwlde on dlfferenL compuLer sysLems lor LhaL
reason lL was programmed Lo run ln MlcrosofL Lxcel (MlcrosofL CorporaLlon) ln whaL follows Lhe four pages of
8LS1 and Lhe sLaLlsLlcal model a alr Wlse llxed 8eallocaLlon 8andomlsaLlon 1esL are descrlbed ln deLall
age 1Introduct|on
Cn Lhe lnLroducLlon page Lhe baslc seLLlngs are made for Lhe 8LS1 appllcaLlon (llg (llg1)1) up Lo four genes and
one reference gene can be labelled ulfferenL background colours ln Lhe spreadsheeLs and Lhe prlnL command are
shown and descrlbed lnk cells lndlcaLe cells for daLa lnpuL blue cells lndlcaLe daLa ouLpuL grey cells are used for
calculaLlon purposes and ouLpuL of Lhe C varlaLlon Lhe red box wlll sLarL Lhe 8andomlsaLlon 1esL lLself and Lhe prlnLer
lcon lndlcaLes 'prlnL Lhls page' lurLher Lhe relaLlve expresslon equaLlon ls glven wlLh dlrecL llnks Lo Lhe daLa lnpuL
secLlon on page C lnpuL + randomlsaLlon LesL
age 2Ck eff|c|ency
1he C8 efflclency calculaLlon ls faculLaLlve and noL obllgaLory for Lhe user (llg (llg2)2) 1o generaLe Lhe daLa basls
for Lhe deLermlnaLlon of C8 efflclency of each LranscrlpL lL ls recommended Lo use varlous dlluLlons ln LrlpleLs of a
pool of all avallable cunAs 1hls ensures Lhe besL esLlmaLlon of Lhe C8 efflclency lf Lhe user wanLs Lo deLermlne Lhe
realLlme C8 efflclencles an lmporL vla copy and pasLe of cunA sLarLlng concenLraLlons ln dlluLlon row and Lhe
correspondlng C values measured by Lhe realLlme C8 machlne ls posslble uependlng on Lhe realLlme C8 plaLform
used C values can be deLermlned elLher by Lhe 1hreshold Cycles llL olnL MeLhod (all plaLforms) or Second
uerlvaLe Maxlmum MeLhod (only LlghLCycler) up Lo Lhree Cs can be lnserLed ln Lhe Lable (run 13) per cunA sLarLlng
concenLraLlon and 8LS1 deLermlnes Lhe slope wlLh a logarlLhmlc algorlLhm as publlshed earller (1614) as well as
an lndlcaLlon of Lhe llnearlLy of Lhls logarlLhmlc allgnmenL uslng earson's correlaLlon coefflclenL 1he realLlme C8
efflclencles were calculaLed from Lhe slope accordlng Lo Lhe esLabllshed equaLlon 10
1/slope
(114) ls ln Lhe range
from 1 (mlnlmum value) Lo 2 (LheoreLlcal maxlmum and opLlmum) lf no realLlme C8 efflclencles are calculaLed here
8LS1 assumes an opLlmal efflclency of 20 on Lhe followlng pages and furLher procedures
age 3C |nput + random|sat|on test
Cn Lhe Lop Lhe calculaLed C8 efflclencles or alLernaLlvely 20 are shown and wlll be Lhe basls for Lhe calculaLlon
and randomlsaLlon LesL (llg (llg3)3) up Lo 16 C daLa per group (conLrol or sample group) can be lnserLed for Lhe
reference gene and up Lo four LargeL genes (lnpuL secLlon of page 3 ls noL shown) Cn cllcklng Lhe red box Lhe
8andomlsaLlon 1esL appllcaLlon wlndow wlll appear Pere Lhe range of Lhe daLa seL musL be deflned for Lhe conLrol
group and sample group by Louchlng Lhe lasL cell conLalnlng Lhe lasL C daLa polnL (on Lhe boLLom rlghL of Lhe plnk
lnpuL wlndow) lurLher Lhe number of randomlsaLlons can be chosen and Lhe randomlsaLlon LesL wlll be sLarLed on
cllcklng Ck lL ls recommended LhaL aL leasL 2000 randomlsaLlons be performed (see nexL secLlon sLaLlsLlcal model)



1he numerlc resulLs of Lhe randomlsaLlon LesL are glven ln Lhe 8andomlsaLlon uaLa CuLpuL box Lhe
concerned Cenes Lhe C mean of conLrol group (ConLrol Means) Lhe C mean of sample group (Sample Means) Lhe
Lxpresslon 8aLlos normallsed by Lhe reference gene Lhe correspondlng pvalues Lhe Lxpresslon 8aLlosnn noL
normallsed by Lhe reference gene Lhe correspondlng pvaluesnn and Lhe number of 8andomlsaLlons performed 1o
slmpllfy maLLers for Lhe user addlLlonal answer senLences were creaLed accordlng Lo Lhe calculaLed resulLs 1hey are
dlvlded lnLo Lhe 8andomlsaLlon 1esL 8esulLs (normallsed by reference gene expresslon) and 8andomlsaLlon 1esL
8esulLs (noL normallsed by reference gene expresslon) 1he senLences Lell Lhe user lf Lhe sample group ln comparlson
wlLh Lhe conLrol group ls up or downregulaLed and lllusLraLes Lhe facLor of regulaLlon and lf Lhls up or down
regulaLlon ls slgnlflcanLly dlfferenL or noL lor upregulaLlon Lhe facLor of regulaLlon ls equal Lo Lhe glven value ln Lhe
8andomlsaLlon uaLa CuLpuL box ln Lhe case of downregulaLlon Lhe regulaLlon facLor ls lllusLraLed as a reclprocal
value (1/expresslon raLlo or 1/expresslon raLlonn respecLlvely)

age 4kat|o + var|at|on output
1he mean C of Lhe genes Lhe C varlaLlons and Lhe coefflclenL of varlaLlon (Cv) are calculaLed and shown Lo lllusLraLe
Lhe reproduclblllLy and varlaLlon of Lhe lnvesLlgaLed group daLa subseLs (llg (llg44)


Stat|st|ca| mode| a|r W|se I|xed kea||ocat|on kandom|sat|on 1est
ulfferences ln expresslon beLween conLrol and LreaLed samples were assessed ln group means (llg (llg1)1) for
sLaLlsLlcal slgnlflcance by randomlsaLlon LesLs (1316 hLLp//wwwblossacuk/smarL/unlx/mrandL/slldes/frameshLm)
ermuLaLlon or randomlsaLlon LesLs are a useful alLernaLlve Lo more sLandard parameLrlc LesLs for analyslng
experlmenLal daLa 1hey have Lhe advanLage of maklng no dlsLrlbuLlonal assumpLlons abouL Lhe daLa whlle remalnlng
as powerful as more sLandard LesLs (16)
1he raLlonale for Lhe randomlsaLlon LesL ls LhaL sLandard parameLrlc LesLs (such as analysls of varlance or tLesLs)
depend on assumpLlons such as normallLy of dlsLrlbuLlons whose valldlLy ls doubLful ln our case where Lhe
quanLlLles of lnLeresL are derlved from raLlos and varlances can be hlgh normal dlsLrlbuLlons would noL be expecLed
and lL ls unclear how a parameLrlc LesL could besL be consLrucLed A randomlsaLlon LesL avolds maklng any
assumpLlons abouL dlsLrlbuLlons and ls lnsLead based on one we know Lo be Lrue LhaL LreaLmenLs were randomly
allocaLed 1he LesL ls conducLed as follows
A sLaLlsLlcal LesL ls based on Lhe probablllLy of an effecL as large as LhaL observed occurrlng under Lhe null hypoLhesls
of no LreaLmenL effecL lf Lhls hypoLhesls ls Lrue Lhe values ln one LreaLmenL group were [usL as llkely Lo have occurred
ln Lhe oLher group 1he randomlsaLlon LesL repeaLedly and randomly reallocaLes Lhe observed values Lo Lhe Lwo
groups and noLes Lhe apparenL effecL (expresslon raLlo ln our case) each Llme 1he proporLlon of Lhese effecLs whlch
are as greaL as LhaL acLually observed ln Lhe experlmenL glves us Lhe lvalue of Lhe LesL
1hey calculaLe lvalues by obLalnlng Lhe proporLlon of random allocaLlons of Lhe mean observed daLa Lo Lhe conLrol
and LreaLed sample groups LhaL would glve greaLer lndlcaLlons of a LreaLmenL effecL Lhan LhaL observed lf Lhls ls small
Lhen Lhere ls evldence LhaL Lhe observed LreaLmenL effecL ls noL slmply Lhe resulL of random allocaLlon 1hus Lhe LesL
makes no assumpLlons concernlng Lhe dlsLrlbuLlon of measured gene expresslon ln any hypoLheslsed populaLlonlL
assumes only Lhe random allocaLlon of LreaLmenL ln pracLlce lL ls lmpracLlcal Lo examlne all posslble allocaLlons of
daLa Lo LreaLmenL groups and a random sample ls drawn lf 2000 or more samples are Laken a good esLlmaLe of Lhe
lvalue (SL 0003 aL l 003) ls obLalned ln Lhe applled alr Wlse llxed 8eallocaLlon 8andomlsaLlon 1esL for each
sample Lhe C values for reference and LargeL genes are [olnLly reallocaLed Lo conLrol and sample groups ( palr wlse
flxed reallocaLlon) and Lhe expresslon raLlos are calculaLed on Lhe basls of Lhe mean values as descrlbed above 1hey
are deemed Lo glve greaLer lndlcaLlons of a LreaLmenL effecL Lhan LhaL acLually observed lf log k log k
0
where
k
0
ls Lhe Lrue expresslon raLlo and k Lhe resulL of reallocaLlon ln Lhe alr Wlse llxed 8eallocaLlon 8andomlsaLlon
1esL a Lwoslded LesL was performed 1he randomlsaLlon LesLs were carrled ouL uslng a MlcrosofL Lxcel macro
(MlcrosofL CorporaLlon) aLLached Lo a purposebullL spreadsheeL and runnlng ln Lhe background of 8LS1

kLSUL1S

Conf|rmat|on of pr|mer spec|f|c|ty
SpeclflclLy of 81C8 producLs was documenLed wlLh hlgh resoluLlon gel elecLrophoresls and resulLed ln a slngle
producL wlLh Lhe deslred lengLh (M1 106 bp CAuP 197 bp) ln addlLlon a LlghLCycler melLlng curve analysls was
performed whlch resulLed ln slngle producLspeclflc melLlng LemperaLures 874C (CAuP) and 897C (M1) no
prlmer prlmerdlmer formaLlons were generaLed durlng Lhe applled 40 realLlme C8 ampllflcaLlon cycles
kea|t|me Ck amp||f|cat|on eff|c|enc|es and var|at|on
8ealLlme C8 efflclencles were calculaLed from Lhe slopes glven ln LlghLCycler sofLware (8oche Molecular
8lochemlcals LlghLCycler SofLware verslon 33) 1he correspondlng realLlme C8 efflclency () of one cycle ln Lhe
exponenLlal phase was calculaLed accordlng Lo Lhe equaLlon 10
1/slope
as descrlbed earller (1614) lnvesLlgaLed
LranscrlpLs showed realLlme C8 efflclency raLes for M1 (
M1
167) and CAuP (
CAuP
188) ln Lhe lnvesLlgaLed
range from 120 pg Lo 73 ng cunA lnpuL repeaLed slx Llmes wlLh hlgh llnearlLy earson correlaLlon coefflclenL (t)
0989
1o mlmlc dlfferenL reverse LranscrlpLlon efflclencles and Lo conflrm preclslon and reproduclblllLy of realLlme C8 as
well as for 8LS1 Lhree repllcaLes of realLlme 81C8 aL each of varlous cunA lnpuL concenLraLlons (Lhree Llmes
more and Lhree Llmes less concenLraLed) were performed and realLlme 81C8 and 8LS1 varlaLlons (Cv) were
deLermlned As shown ln 1able 1able11 varlaLlons of lnvesLlgaLed LranscrlpLs are based on Lhe C varlaLlon and
remalned sLable beLween 243 and 1003 for M1 and 139 and 1289 for CAuP Lhe laLLer showlng a dependence
on Lhe cunA lnpuL ln realLlme C8 C lLself decreased wlLh lncreaslng cunA lnpuL ln boLh facLors and groups
Var|at|on and reproduc|b|||ty of kLS1
Cn Lhe basls of Lhe prevlously publlshed maLhemaLlcal model (6) 8LS1 calculaLes Lhe relaLlve expresslon raLlos on
Lhe basls of group means for LargeL gene M1 versus reference gene CAuP and LesLs Lhe group raLlo resulLs for
slgnlflcance normallsed and noLnormallsed expresslon resulLs were compared
-tmollseJ by CAlun exptesslo As presenLed ln 1able 1able22 Lhe downregulaLlon facLor (reclprocal value of
raLlo) of M1 m8nA ln Lhe case of zlnc deflclency was calculaLed by 8LS1 sLarLlng from dlfferenL cunA concenLraLlons
lurLher dlfferenL runs (M1 13 and CAuP 13 o 3 9) were compared Lo calculaLe all posslble comblnaLlons
beLween lndlvldual realLlme runs uerlved varlaLlons and Lhe lnfluence of devlaLlng cunA sLarLlng amounLs on Lhe
8LS1 calculaLed relaLlve expresslon raLlo and Lhe slgnlflcance of Lhe performed randomlsaLlon LesL are presenLed ln
1able 1able22 Cver all lnvesLlgaLed comblnaLlons (o 27) a mean facLor of downregulaLlon of 44303 (Cv 2683)
was observed no slgnlflcanL dlfferences beLween cunA sLarLlng concenLraLlon on expresslon raLlo could be found


Table 2
Factor oI down-regulation oI MT versus GAPDH expression levels in rat liver under zinc depletion (
normalised by the GAPDH expression)


Lxpressed facLor of downregulaLlon (1/raLlo) and lvalues of conLrol group (o 7) versus zlnc depleLlon (sample
group o 6) were calculaLed by 8LS1 uaLa were deLermlned ln LrlpleLs aL each of dlfferenL sLages of cunA lnpuL
(823 23 and 73 ng) accordlng Lo Lhe C values glven ln 1able 1 AnCvA (o 3 9) was performed Lo LesL Lhe
lnfluence of cunA sLarLlng concenLraLlon on expresslon raLlo lncludlng normallsaLlon by CAuP 1here was no
slgnlflcanL dlfference beLween concenLraLlon buL a hlghly slgnlflcanL effecL beLween M1 and especlally beLween
CAuP repllcaLes
- otmollsotlo by CAlun ln 1able 1able33 Lhe facLor of downregulaLlon of M1 m8nA ln Lhe case of zlnc deflclency
was calculaLed by 8LS1 wlLhouL normallsaLlon by Lhe reference gene lor M1 a mean downregulaLlon facLor of
28081 (Cv 1022) and for CAuP of 0677 (Cv 2979) were observed no slgnlflcanL dlfferences beLween cunA
sLarLlng concenLraLlon on expresslon raLlo could be found elLher for M1 or CAuP

DISCUSSICN

1oday realLlme 81C8 uslng fluorescence dyes slgnlflcanLly slmpllfles and acceleraLes Lhe process of produclng
reproduclble and rellable quanLlflcaLlon of m8nA (1) 1hls has led Lo Lhe developmenL of new klneLlc 81C8
meLhodologles LhaL are revoluLlonlslng Lhe posslblllLles of m8nA quanLlflcaLlon (17) AbsoluLe quanLlflcaLlon ls very
common where an approprlaLe exLernal callbraLlon curve ls used Lo deLermlne Lhe absoluLe m8nA copy number (2)
Cn Lhe oLher hand relaLlve expresslon wlll be lncreaslngly performed by researchers accordlng Lo several esLabllshed
maLhemaLlcal models (68) 8uL unLll now no rellable appllcaLlon was avallable for a groupwlse calculaLlon of Lhe
relaLlve expresslon raLlo and a subsequenL sLaLlsLlcal comparlson of Lhe resulLs by a sLaLlsLlcal LesL Pereln a new
sofLware Lool ls presenLed and descrlbed whlch allows for such a group comparlson and sLaLlsLlcal analysls 8LS1 ls
based on an efflclency correcLed maLhemaLlcal model for daLa analysls lL calculaLes Lhe relaLlve expresslon raLlo on
Lhe basls of Lhe C8 efflclency () and crosslng polnL devlaLlon (AC) of Lhe lnvesLlgaLed LranscrlpLs (6) and on a newly
developed randomlsaLlon LesL macro
Cross|ngpo|nt determ|nat|on
lor Lhe deLermlnaLlon of C ln general Lwo meLhods can be chosen Lhe llL olnL MeLhod or adequaLe meLhodologles
llke 1hreshold Cycle (1819) where C wlll be measured aL consLanL fluorescence level and Lhe Second uerlvaLlve
Maxlmum MeLhod where C wlll be measured aL Lhe maxlmum lncrease or acceleraLlon of fluorescence even lf Lhe
fluorescence levels beLween curves are dlfferenL (14) 8esldes Lhe LlghLCycler Lhe llL olnL MeLhod or 1hreshold Cycle
are used ln 1aqMan (L Applled 8losysLems losLer ClLy CA) 8oLoCene (CorbeLL 8esearch Sydney AusLralla)
lCycler 1hermal Cycler (8lo8ad Percules CA) and MulLlplex CuanLlLaLlve C8 SysLem (SLraLagene La !olla CA) 1he
Second uerlvaLe Maxlmum MeLhod ls an algorlLhm excluslvely used ln LlghLCycler sofLware (8oche Molecular
8lochemlcals LlghLCycler SofLware verslon 33)
Norma||sat|on
1he normallsaLlon of Lhe LargeL gene wlLh an endogenous sLandard ls recommended 8LS1 allows for a
normallsaLlon of Lhe LargeL genes wlLh a reference gene Cn boLh maLhemaLlcal models Lhe alr Wlse llxed
8eallocaLlon 8andomlsaLlon 1esL ls performed and Lhe resulLs are presenLed ln Lhe approprlaLe ouLpuL wlndows
8esearchers can declde lf Lhey wanL Lo correcL Lhe daLa or noL 1he basls of daLa normallsaLlon ls Lhe expresslon resulL
of an endogenous deslrable unregulaLed reference gene LranscrlpL Lo compensaLe lnLerC8 varlaLlons (sample Lo
sample varlaLlons) beLween Lhe runs lf Lhe C devlaLlon of Lhe chosen reference gene has Lhe same mean ln Lhe
conLrol as ln sample group mean ACref
(mean conLrol mean sample)
0 Lhen a sLable and consLanL
reference gene m8nA level ls glven 8ealLlme 81C8speclflc errors ln Lhe quanLlflcaLlon of m8nA LranscrlpLs are
easlly compounded wlLh any varlaLlon ln Lhe amounL of sLarLlng maLerlal beLween Lhe samples 1hls ls especlally
relevanL when Lhe samples have been obLalned from dlfferenL lndlvlduals and wlll resulL ln Lhe mlslnLerpreLaLlon of
Lhe derlved expresslon proflle of Lhe LargeL genes (1) Pere some quesLlons arlse whaL ls Lhe approprlaLe reference
gene for an experlmenLal LreaLmenL and lnvesLlgaLed Llssue (1120)? Commonly used housekeeplng genes (9) are
sulLable for reference genes slnce Lhey are presenL ln all nucleaLed cell Lypes and necessary for baslc cell survlval 1he
m8nA synLhesls of housekeeplng genes ls consldered Lo be sLable ln varlous Llssues even under experlmenLal
LreaLmenLs (911) 8uL numerous LreaLmenLs and sLudles have already shown LhaL housekeeplng genes are regulaLed
and vary under speclflc experlmenLal condlLlons (2124) 1hls ls a fundamenLal problem for each relaLlve
quanLlflcaLlon and correcLlon or normallsaLlon ln nuclelc acld based models glven ln array experlmenLs as well as ln
m8nA expresslon analysls lf one deslred reference ls regulaLed ln a speclflc experlmenLal Lrlal lL remalns Lo Lhe
lnvesLlgaLor Lo declde whlch gene can flL Lhe hypoLhesls of a nonregulaLed reference for a rellable normallsaLlon
1herefore he has Lo LesL for more housekeeplng genes and calculaLe a Pousekeeplng Cene lndex (publlcaLlon ln
preparaLlon) Accordlng Lo Lhls Pousekeeplng Cene lndex whlch ls based on Lhe expresslon of aL leasL Lhree
housekeeplng genes a more rellable basls of normallsaLlon ln relaLlve quanLlflcaLlon uslng 8LS1 can be posLulaLed
1he endogenous conLrol or Lhe calculaLed Pousekeeplng Cene lndex should be expressed aL roughly Lhe same C
range as Lhe LargeL gene (1) ln same C range reference and LargeL underwenL already Lhe same cycle condlLlon real
Llme 81C8 klneLlcs wlLh respecL Lo polymerase acLlvaLlon (heaL acLlvaLlon of polymerase) or lnacLlvaLlon and
reacLlon end producL lnhlblLlon by Lhe generaLed 81C8 producL (23) 8LS1 can glve you Lhe flrsL essenLlal hlnLs lf a
normallsaLlon vla Lhe chosen reference gene ls useful (by Lhe facLor of regulaLlon and pvaluenn of Lhe randomlsaLlon
LesL of Lhe reference) or lf Lhe reference ls noL sulLable because lL ls slgnlflcanLly regulaLed
Lff|c|ency correct|on
8eslde Lhe normallsaLlon by a reference Lhe C8 efflclency ln realLlme C8 has a ma[or lmpacL on Lhe accuracy of Lhe
calculaLed expresslon resulL (8oche Molecular 8lochemlcals LlghLCycler 8elaLlve CuanLlflcaLlon SofLware verslon 10)
A correcLlon for efflclency as performed ln equaLlons 1 and 2 ls recommended and resulLs ln a more rellable
esLlmaLlon of Lhe 'real' expresslon raLlo compared wlLh Lhe no efflclency correcLlon Small efflclency dlfferences
beLween LargeL and reference genes generaLe false expresslon raLlo and Lhe researcher over or underesLlmaLes Lhe
'real' lnlLlal 8nA amounL When Lhe dlfference (A) ln C8 efflclency () ls A 003 beLween LargeL and reference gene
Lhe falsely calculaLed dlfference ln expresslon raLlo ls 46 ln Lhe case of
LargeL

ref
and 209 ln Lhe case of

LargeL

ref
afLer 23 performed cycles 1hls dlfference wlll lncrease dramaLlcally by hlgher efflclency dlfferences A
003 (27 and 338) and A 010 (72 and 1083) and hlgher cycles performed 1herefore efflclency correcLed
quanLlflcaLlon ls calculaLed auLomaLlcally by 8LS1 based on Lhe meLhod descrlbed on page 2 (llg (llg2)2) lL ls
recommended Lo perform Lhe deLermlnaLlon of realLlme C8 efflclency ln LrlpleLs for every Llssue separaLely ln a pool
of all sLarLlng 8nAs Lo accumulaLe all posslble lmpacLs on C8 efflclency As ls known each Llssue exhlblLs an lndlvldual
C8 efflclency caused by 81 and C8 lnhlblLors (purlfled ln 8nA exLracLlon) and by varlaLlons ln Lhe LoLal 8nA paLLern
exLracLed
ke|at|ve quant|f|cat|on software
up Lo now only one relaLlve quanLlflcaLlon sofLware program for realLlme C8 has been avallable and ls dlsLrlbuLed by
8oche Molecular 8lochemlcals Lhe LlghLCycler 8elaLlve CuanLlflcaLlon sofLware (verslon 10 8oche Molecular
8lochemlcals) 1he maLhemaLlcal algorlLhm on whlch Lhe 8oche Molecular 8lochemlcals sofLware ls based ls
unpubllshed and mlghL be Lhe one dlscussed earller (68)
8aLlo (
ref
)
C
sample
/(
LargeL
)
Csample
/(
ref
)
C
callbraLor
/ (
LargeL
)
C
callbraLor
2
1he LlghLCycler 8elaLlve CuanLlflcaLlon sofLware allows only for a comparlson of maxlmal LrlpleLs (o 3) of a LargeL
versus a callbraLor (cal) gene (whlch ls ldenLlcal Lo Lhe conLrol) boLh correcLed vla a reference (ref) 1he relaLlve and
normallsed expresslon raLlo ls calculaLed on Lhe basls of Lhe medlan of Lhe performed LrlpleLs and compuLed accordlng
Lo Lhe glven equaLlon 3 (8oche Molecular 8lochemlcals LlghLCycle 8elaLlve CuanLlflaLlon SofLware verslon 10) 1hls
equaLlon conLalns a correcLlon facLor (Cl) as well as a mulLlpllcaLlon facLor (Ml) whlch are provlded ln Lhe producL
speclflc appllcaLlons by 8oche Molecular 8lochemlcals 8aLlo concenLraLlon (conc) are derlved from relaLlve sLandard
curves uslng Lhe C medlan values 1argeL Lo reference raLlos of all samples are referenced Lo Lhe LargeL Lo reference
raLlo of Lhe callbraLor 1hus lL ls lmporLanL Lo correcL for loLLoloL dlfferences of Lhe callbraLor for comparablllLy of
daLa (8oche Molecular 8lochemlcals LlghLCycle 8elaLlve CuanLlflaLlon SofLware verslon 10)
8aLlo conc(LargeL sample)/conc(reference sample) * Ml/
conc(LargeL callbraLor/conc(reference callbraLor) * Cl 3
Advantages of kLS1
8LS1 allows a comparlson of four LargeL genes wlLh a reference gene ln Lwo experlmenLal groups wlLh up Lo 16 daLa
polnLs per group 8elaLlve quanLlflcaLlon of a LargeL LranscrlpL ls based on Lhe mean C devlaLlon of conLrol and
sample group normallsed by a reference LranscrlpL 8ealLlme C8 efflclency correcLlon can be performed and ls
hlghly recommended normallsaLlon vla an endogenous sLandard can be performed accordlng Lo Lhe users demand
buL lL ls recommended Lo compensaLe lnLer81C8 (or sample Lo sample) varlaLlons (8oche Molecular 8lochemlcals
LlghLCycle 8elaLlve CuanLlflaLlon SofLware verslon 10) varlaLlons ln 8nA lnLegrlLy 81 efflclency dlfferences and cunA
sample loadlng varlaLlons (26) 1herefore a hlgh reproduclblllLy of 81 and 81 efflclency whlch greaLly varles beLween
Llssues Lhe applled 8nA lsolaLlon meLhodology and Lhe 81 enzymes used (2728) are noL lmporLanL any more Pereln
dlfferenL cunA lnpuL concenLraLlons were LesLed (300) Lo mlmlc Lhese huge 81 varlaLlons and resulLed ln no
slgnlflcanL changes of relaLlve expresslon raLlo evaluaLed by 8LS1 Also Lhe reproduclblllLy of Lhe developed
maLhemaLlcal model used ln 8LS1 was glven based on Lhe exacL deLermlnaLlon of realLlme ampllflcaLlon
efflclencles and low LlghLCycler C varlablllLy documenLed ln 8LS1
a|r W|se I|xed kea||ocat|on kandom|sat|on 1est
8andomlsaLlon LesLs wlLh a palrwlse reallocaLlon were seen as Lhe mosL approprlaLe approach for Lhls appllcaLlon
1hey make no assumpLlons abouL Lhe dlsLrlbuLlon of observaLlons ln populaLlons whlch would always be quesLlonable
for gene expresslon measuremenLs lnsLead Lhey assume LhaL anlmals were randomly allocaLed Lo conLrol and
LreaLmenL groups whlch ls known Lo be Lrue lf Lhe experlmenLal proLocol was adhered 1hey are more flexlble Lhan
nonparameLrlc LesLs based on ranks (MannWhlLney kruskalWallls eLc) and do noL suffer a reducLlon ln power
relaLlve Lo parameLrlc LesLs (tLesLs AnCvA eLc) 1hey can be sllghLly conservaLlve (le Lype l error raLes lower Lhan
Lhe sLaLed slgnlflcance level) due Lo accepLance of randomlsaLlons wlLh group dlfferences ldenLlcal Lo LhaL observed
buL Lhls malnly occurs when used wlLh dlscreLe daLa (whlch gene expresslon daLa are noL) and small sample slzes

COCUSOS

REST using the Pair Wise Fixed Reallocation Randomisation Test is presented Ior a better understanding
oI relative quantiIication analysis in real-time RTPCR. In rat liver the MT down-regulation in the zinc
deIiciency group versus the control group lead to similar results using either a normalisation or no
normalisation via GAPDH. Real-time RTPCR in combination with REST is the method oI choice Ior any
experiments requiring sensitive, speciIic and reproducible quantiIication oI mRNA. The soItware developed,
based on the described mathematical model, exhibits suitable reliability as well as reproducibility in
individual runs, conIirmed by high accuracy and low variation independent oI huge template concentration
variations. The latest version oI REST and examples Ior the correct use can be downloaded at
http://www.wzw.tum.de/gene-quantiIication/.

CKOWEDGEETS

The author thanks D. Schmidt Ior technical assistance. The experimental trial was perIormed in collaboration
with the Animal Nutrition and Production Physiology, Center oI LiIe and Food Sciences, Technical
University oI Munich, under the supervision oI Dr W. Windisch.

kLILkLNCLS

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ArLlcles from nuclelc Aclds 8esearch are provlded here courLesy of
Cxford Un|vers|ty ress


Source: hffp://www.ncbi.nIm.nih.gov/pmc/orficIes/PMCII38b9/
DownIood by: AbdeIhody Abdeen
Coiro in: Sofurdoy, Sepfember I7, Z0II
b:3Z PM