livak_schmittgen2001

8/20/2019 Livak_Schmittgen2001

http://slidepdf.com/reader/full/livakschmittgen2001 1/7

METHODS 2 5 , 402–408 (2001)

doi:10.1006/ meth.2001.1262, available online at http:/ / www.idealibrary.com on

Analysis of Relative Gene Expression Data Using Real-

Time Quantitative PCR and the 2

C T

MethodKenneth J . Livak* a nd Thom a s D. S chm itt gen† ,1

*A ppli ed Bi osystems, Foster City, Cali forn ia 94404; and † Department of Pharmaceutical Sciences, College of Pharmacy,

Washin gton State Un iversity, Pul lm an, Washi ngton 99164-6534

of t h e t a r g et g en e r e la t i ve t o s o m e r e f er e n ce g r ou pThe two most commonly used methods to analyze data from

s u ch a s a n u n t rea t ed con t ro l or a s a mp le a t t ime z eroreal-time, quantitative PCR experiments are absolute quantifica-

in a time-course study.tion and relative quantification. Absolute quantification deter-

Absolute q ua ntificat ion should be performed in s itu-mines the input copy number, usually by relating the PCR signalto a standard curve. Relative quantification relates the PCR signal a t ions wh ere it is necessa ry to determine the absoluteof the target transcript in a treatment group to that of another t ra n s crip t c op y n u mb er. Ab s olu t e q u a n t i f ica t ion h a ssample such as an untreated control. The 2C T method is a b een comb in ed w it h rea l-t ime P CR a n d n u m erou s re-convenient way to analyze the relative changes in gene expression

ports have appeared in the literature (6– 9) includingfrom real-tim e quantit ative PCR experiments. The purpose of this

tw o ar t icles in t his issue (10, 11). In some sit ua tions,report is to present the derivation, assumptions, and applicationsi t ma y b e u n n eces sa r y t o d et ermin e t h e a b s o lu t e t ra n -of the 2C T method. In addition, we present the derivation andscript copy number and reporting the relat ive changeapplications of two variations of the 2C T method that may be

useful in the analysis of real-tim e, quantitat ive PCR data. 2001 in gen e expres s ion w il l s u f fice. F or exa m p le, s t a t in gElsevier Science (USA) t h a t a g i ve n t r e a t m e n t i n cr e a s ed t h e e xp r es s ion of

Key Words: reverse transcription polymerase chain reaction; gene x b y 2. 5-fold ma y b e mo re relev a n t t h a n s t a t in gquantitative polymerase chain reaction; relative quantification;

t h a t t h e t rea t men t in c rea s ed t h e expres s ion o f gen e x real-time polymerase chain reaction; Taq Man.from 1000 copies to 2500 copies per cell.

Qua nt ifying the rela t ive cha nges in gene expression

u s in g r e a l -t i m e P C R r e q u ir e s c er t a i n e q u a t i on s , a s -

s u m p t i o n s , a n d t h e t e s t i n g o f t h e s e a s s u m p t i o n s t oRes erv e t ra n s crip t ion comb in ed w it h t h e p oly mer- p rop erly a n a ly z e t h e d a t a . Th e 2C T met h o d ma y b e

a se cha in rea ction (RT-P CR ) ha s proven to be a power- used to calculate rela t ive cha nges in gene expressionfu l m et h od t o q u a n t i fy gen e expres s ion (1– 3). Rea l- d et ermin ed fro m rea l- t ime q u a n t i t a t iv e P CR exp eri-t i m e P C R t e c h n o l o g y h a s b e e n a d a p t e d t o p e r f o r m m e n t s . D e r i v a t i o n o f t h e 2C T eq u a t ion , in clu d in gqua ntit a t ive RT-P CR (4, 5). Two different meth ods of a s s u m p t ion s , e xp er i m en t a l d es i gn , a n d v a l i da t i ona n a l y zi n g d a t a f r om r e a l -t i m e, q u a n t i t a t i v e P C R e x- tests, have been described in Applied Biosystems User

p erimen t s exis t : a b s o lu t e q u a n t i f ic a t io n a n d rela t iv e B ullet in N o. 2 (P /N 4303859). Ana lys es of gene expr es-qua ntificat ion. Absolute qua nt ifica t ion determin es th es ion d a t a u s in g t h e 2C T met h o d h a v e a p p ea red in

input copy number of th e tr a nscript of int erest , usua llythe literature (5, 6). The purpose of this report is to

b y r e la t i n g t h e P C R s i g n a l t o a s t a n d a r d cu r v e. R el a -present the derivat ion of the 2C T met h o d , a s s u mp -tive qua ntificat ion describes the cha nge in expressiont ion s in v olv ed in u s in g t h e met h o d , a n d a p p lica t ion s

of t h is m et h od fo r t h e gen era l l i t era t u re. In a d d it ion ,

we present t he derivat ion and a pplica t ion of two varia -

t ion s o f t h e 2C T m e t h od t h a t m a y b e u se fu l i n t h e1 To whom requests for r eprints should be a ddressed. Fa x: (509)335-5902. E-mail: [email protected]. a n a l y si s of r e a l -t i m e q u a n t i t a t i v e P C R d a t a .

402 1046-2023/01 $35.00

2001 E lsevier S cience (USA)All rights reserved.



ANALYSIS OF REAL-TIME PCR DATA 403

or1. THE 2C T METHOD

X N (1 E )C T K , [6]

1.1. Derivation of t he 2C T Methodw h ere X N i s eq u a l t o t h e n o rma liz ed a mou n t of t a rgetThe equation that describes the exponential amplifi-(X 0 /R 0) a n d C T is equal to th e difference in thresholdcation of PCR iscycles for ta rget a nd reference (C T,X C T,R ).

Rearr an ging gives th e expressionX n X 0 (1 E X)n , [1]

X N K (1 E )C T. [7]

w h ere X n i s t h e n u mb er o f t a rget mo lec u les a t c y c leThe fina l s t ep is to divide th e X N for a n y s a mp le q byn of the reaction, X 0 i s t h e i n i t i a l n u m b e r o f t a r g e tt h e X N for th e calibra tor (cb):molecules. E X is t he efficiency of ta rget am plifica t ion,

a n d n is the number of cycles. The threshold cycle (C T)

i n di ca t e s t h e f r a ct i on a l cy cl e n u m b er a t w h i ch t h e X N, q

X N,cb

K (1 E )C T,q

K (1 E )C T,cb (1 E )C T. [8]

amount of amplified target reaches a f ixed threshold.

Thus,

H ere C T (C T,q C T,cb).

For a mplicons designed to be less tha n 150 bp an d forX T

X 0

(1

E X)C T,X

K X [2] w h ic h t h e p rimer a n d M g 2+ concentra t ions ha ve been

properly optim ized, t he efficiency is close to one. There-w h ere X T is the threshold number of target molecules, fore, th e a mount of ta rget, norma lized to a n endogenousC T,X is the threshold cycle for t ar get a mplificat ion, a nd reference a nd r ela t ive to a ca libra tor, is given byK X is a const a nt . A simila r equa tion for th e endogenous

reference (intern a l cont rol gene) reaction is amount of target 2C T. [9]

R T R 0 (1 E R)C T,R K R, [3]1.2. Assumptions and Applications of the 2C T Method

For th e C T calculat ion to be va lid, th e a mplifica tionw h ere R T is the threshold number of reference mole-efficiencies of th e ta rget a nd r eference must be approxi-cules, R 0 is the initial number of reference molecules,

mately equal. A sensit ive method for assessing if twoE R is the efficiency of reference amplification, C T,R isamplicons have the same efficiency is to look at how the threshold cycle for reference amplification, and K RC T va ries with template dilution. Figure 1 shows theis a constant .

Dividing X T b y R T gives t he expression

X T

R T

X 0 (1 E X)C T,X

R 0 (1 E R)C T,R

K X

K R K . [4]

For rea l-time a mplifica tion using Ta qMa n probes, th e

exact va lues of X T a n d R T depend on a number of factors

including the reporter dye used in the probe, the se-quence context effects on the fluorescence properties of

the probe, the efficiency of probe cleavage, purity of

the probe, and sett ing of the fluorescence threshold.

Therefore, t he consta nt K does not ha ve to be equa l toFIG. 1. V alidat ion of t he 2C T method: Amplification of cDNAone. Assuming efficiencies of the t ar get a nd the refer-synt hesized from different a mounts of RNA. The efficiency of amplifi-

en ce a re t h e s a me,cation of the target gene (c-m yc ) and int ernal cont rol (GAPD H) w a sexamined using real-time P CR a nd Ta qMa n detection. Us ing reverset ra nscript a se, cDNA w a s synt hesized from 1 g t ot al R NA isolat edE X E R E ,from human Raji cells. Serial dilutions of cDNA were amplified byreal-time PCR using gene-specific primers. The most concentratedsample contained cDNA derived from 1 ng of total RNA. The C T(C T,cmy c C T,GAP DH ) wa s calculated for each cDNA dilution. The da ta

X 0

R 0 (1 E )C T,XC T,R K , [5]

w ere f i t u sing least -squ ares l inear regression ana lysis (N 3).



LIVAK AND SCHMITTGEN404

results of a n experiment where a cDNA prepara t ion cha nge in gene expression relat ive to an untreat ed con-

tr ol, for exam ple, if one wa nt ed to determ ine the expres-wa s diluted over a 100-fold ra nge. For ea ch dilution

sam ple, a mplifica t ions w ere performed using primers s ion of a part icular mRNA in an orga n. In these cases,

the calibra tor ma y be the expression of the sam e mRNAa nd flu orogenic probes for c-m yc a nd G AP DH . The aver-

a ge C T wa s calculat ed for both c-m yc a n d G A P D H a n d i n a n ot h e r o rg a n . Ta b l e 1 p r es en t s m e a n C T values

determined for c-m yc a n d G A P DH t ra n s c rip t s in t o t a lt h e C T (C T,m yc C T,G APD H ) wa s determined. A plot of

the log cDNA dilution versus C T w a s ma d e (F ig. 1). RN A s a mp les from b ra in a n d k id n ey. Th e b ra in w a s

a rbitr a rily chosen a s the ca libra tor in this exa mple. TheIf the absolute value of the slope is close to zero, theefficiencies of the ta rget a nd reference genes a re s imi- am ount of c-m yc , norma lized to G AP DH a nd relat ive to

b ra in , is report ed . A lt h o ugh t h e rela t iv e q u a n t i t a t iv elar, a nd the C T calculation for the relative quantifica-

tion of ta rget ma y be used. As shown in Fig. 1, th e slope meth od can be used to ma ke th is ty pe of tissue compari-

son, biological int erpreta tion of th e results is complex.of the line is 0.0471; therefore, t he a ssumption holds

a n d t h e C T met h od ma y b e u s ed t o a n a ly z e t h e d a t a . Th e s in gle rela t iv e q u a n t i t y report ed a ct u a l ly reflect s

va ria tion in both ta rget a nd reference tr a nscripts a crossIf the efficiencies of the two amplicons are not equal,

t h e n t h e a n a l y si s m a y n ee d t o be p er f or m ed v i a t h e a v a r i et y of c el l t y pe s t h a t m i gh t b e pr es en t i n a n y

part icular t issue.a bsolute qua ntificat ion meth od using sta nda rd curves.

Alter na tively, new pr imer s ca n be designed a nd/or opti-

mized to achieve a s imila r efficiency for the ta rget a nd1.4. Data Analysis Using the 2C T Methodreference a mplicons.

Th e C T values provided from real-t ime PCR instru-

menta t ion a re easily imported into a spreadsheet pro-1.3. Selection of Internal Control and Calibrator for the gra m such a s Microsoft E xcel. To demonstr a te th e an a l-2C T Method y sis , d a t a a r e r epor t ed f rom a q u a nt it a t iv e g en e

expression experiment a nd a sa mple sprea dsheet is de-The purpose of th e intern a l cont rol gene is to norma l-

ize t he P CRs for the a mount of RNA a dded to t he re- scribed (Fig. 2). The change in expression of the f os–gl o–

m yc t a rget gen e n orma liz ed t o -actin wa s m onitoredv er s e t r a n s cr i pt i on r ea c t ion s . We h a v e f ou n d t h a t

sta nda rd housekeeping genes usually suffice as int er- over 8 h. Triplica te sa mples of cells were collected a t

each t ime point . Rea l-t ime P CR wa s performed on thenal control genes. Suitable internal controls for real-

t i m e q u a n t i t a t i v e P C R i n c l u d e G A P D H , -actin, 2- corresp on d in g c DN A s y n t h es iz ed from ea ch s a mp le.

The data were analyzed using Eq. [9], where C T microglobulin, a nd rRNA. Other housekeeping genes

w i ll u n dou bt ed ly w or k a s w e ll . I t i s h i gh ly r ecom - (C T,Ta rg et C T,Act in )Time x (C T,Ta rg et C T,Act in )Time 0. Timex is any t ime point a nd Time 0 represents th e 1 expres-mended that the internal control gene be properly vali-

da ted for each experiment t o determine th a t gene ex- s ion of the ta rget gene normalized to -a ctin. The mea n

C T values for both th e ta rget a nd interna l control genespression is unaffected by the experimental treatment.

A met h od t o v a l id a t e t h e effect o f experimen t a l t rea t - w ere d et ermin ed a t t ime z ero (F ig. 2 , colu mn 8) a n d

w ere used in Eq . [9]. The fold cha nge in t he ta rget gene,ment on the expression of t he interna l control gene is

descr ibed in S ect ion 2.2. n orm a lized t o -actin and relative to the expression at

time zero, was calculated for each sample using Eq. [9]The choice of calibrator for the 2C T method de-

p en d s on t h e t y p e of gen e express ion exp erimen t t h a t (F ig. 2 , c olu mn 9). Th e mea n , S D , a n d CV a re t h en

d et ermin ed fro m t h e t r ip l ic a t e s a mp les a t ea c h t imeone has planned. The simplest design is to use the un-

t r e a t e d c on t r ol a s t h e ca l i br a t o r. U s i n g t h e 2C T p oin t . U s in g t h is a n a ly s is, t h e v a lu e o f t h e m ea n fold

change at t ime zero should be very close to one (i .e. ,met h od , t h e d a t a a re p res en t ed a s t h e fold c h a n ge ingene expression norma lized to a n endogenous reference since 2 0 1). We h a ve found the verifica t ion of the

mea n fold cha nge at tim e zero to be a convenient meth odgene and relat ive to the unt reat ed control. For t he un-

treated control sample, C T equals zero an d 20 eq u a ls t o ch eck for errors a n d v a ria t ion a m on g t h e t r ipl ica t e

samples. A value that is very different from one sug-one, so tha t t he fold chan ge in gene expression rela tive

to the untr eated control equals one, by definit ion. For gests a calculat ion error in the sprea dsheet or a very

high degree of experimenta l va riat ion.the t reat ed samples, evalua tion of 2C T indicat es the

fold change in gene expression relat ive to the untrea ted In the preceding example, three separ at e RNA prepa -

r a t i on s w e r e m a d e f or e a ch t i m e p oi n t a n d ca r r i edcontr ol. Similar a na lysis could be a pplied t o s tudy t he

time course of gene expression where the ca libra tor through the an a lysis. Therefore, i t ma de sense to trea t

ea c h s a mp le s ep a ra t ely a n d a v era ge t h e res u lt s a f t ersam ple represents the a mount of tra nscript tha t is ex-

pr essed a t t im e zer o. t h e 2C T calculat ion. When replicate PCRs are run

on t he same sa mple, i t is more appropriate t o averageS i t u a t i on s e xi st w h e r e o ne m a y n ot com pa r e t h e




C T data before performing the 2C T ca lcu la t ion . E x- (G AP DH ) w ere a mp lif ied in s ep a ra t e w ells . Th ere is

n o rea s o n t o p a ir a n y p a rt ic u la r c -m yc w e ll w i t h a n yactly how the averaging is performed depends on if the

t a rget a n d referen ce a re a mp li fied in s ep a ra t e w ells p a rt icu la r G AP DH w ell. Th erefore, i t ma k es sen se t o

a v era ge t h e c -m yc a n d G AP D H C T values separatelyor i n t h e s a m e w e ll . Ta b l e 1 p r e se nt s d a t a f r om a n

exp erimen t w h ere t h e t a rget (c-m yc ) a n d r e fe re nce b ef or e pe rf or m in g t h e C T calculat ion. The variance

TABLE 1

Trea tment of Replica te D a ta Where Tar get a nd Reference Are Amplified in Sepa ra te Wellsa

C T (Avg. c-myc C T C T (Avg. C T Normalized c-m yc a mountTissue c-myc C T G A P D H C T Avg. G AP DH C T Avg. C T,Br ain ) rela tive t o bra in 2C T

B r a in 30.72 23.7030.34 23.5630.58 23.4730.34 23.6530.50 23.6930.43 23.68

Aver a ge 30.49 0.15 23.63 0.09 6.86 0.17 0.00 0.17 1.0 (0.9– 1.1)

K idney 27.06 22.7627.03 22.6127.03 22.6227.10 22.6026.99 22.6126.94 22.76

Aver a ge 27.03 0.06 22.66 0.08 4.37 0.10 2.50 0.10 5.6 (5.3– 6.0)

a Tot al R NA from hu ma n brain and kidney w ere p u rchased from Clont ech. U sing reverse t ra nscrip t ase, cDNA w as synt hesized from 1 g tota l RNA. Aliquots of cDNA were used a s templa te for real-time P CR rea ctions containing either primers a nd probe for c-m yc or primersand probe for GAPDH. Each reaction contained cDNA derived from 10 ng total RNA. Six replicates of each reaction were performed.

FIG. 2. Samp le sp readsheet of dat a analysis u sing t he 2C T method. The fold change in expression of the target gene ( fos–gl o–m yc )relative to the internal control gene ( -act in) at various t ime p oint s w as st u died. The sa mp les w ere a nalyzed u sing rea l-t ime qu a nt it at iveP C R a n d t h e C t data were imported into Microsoft Excel. The mean fold change in expression of the target gene at each time point wascalculated using Eq . [9], w here C T (C T,Tar ge t C ,Actin)Time x (C T,Tar ge t C ,Actin)Time 0. The mea n C T at time zero are shown (colored boxes)

as is a sample calculation for the fold change using 2C T (black box).




e st i m a t e d f r om t h e r e pl ica t e C T v a lu es is ca rr ied of a n a r b it ra ry con s t a n t . Th is gives resu lt s eq u iv a len t

to those report ed in Fig. 2 where C T values for nonrepli-t h ro ugh t o t h e f in a l ca lc ula t ion of rela t iv e q u a n t i t ies

using sta nda rd propaga tion of error methods. One diffi- cat ed sa mples w ere car ried th rough the entire 2C T

calculation before averaging. Alternatively, it is possi-culty is tha t C T is exponentia lly rela ted t o copy nu mber

(see Section 4 below ). Thus, in the final calculation, the ble to report results w ith the calibra tor qua ntit y defined

a s 1 w i t h ou t a n y error . In t h is c a s e, t h e erro r es t i-error is est ima ted by evaluat ing the 2C T term using

C T p lu s t h e s t a n d a rd d evia t ion a n d C T m i n us t h e m a t e d f or t h e a v e r a g e C T,cb value must be propaga ted

in t o ea ch of t h e C T v a lu es for t h e t est s a mp les . Insta nda rd deviat ion. This lea ds to a ra nge of values tha tis a s y mmet rica l ly d is t r ib u t ed rela t iv e t o t h e a v era ge Ta b le 1, t h e C T value for the kidney sample would

become 2.50 0.20 an d th e norma lized c-m yc amountva lue. The a symm etric distr ibution is a consequence of

converting the results of an exponential process into a would be 5.6 with a range of 4.9 to 6.5. Results for

brain would be reported as 1 without any error.linear compar ison of a mounts .

B y using probes labeled with dist inguisha ble report er

dyes, it is possible to run t he ta rget a nd reference ampli-

ficat ions in the sa me w ell. Table 2 presents dat a from 2. THE 2C T METHODan experiment where the target (c-m yc ) an d reference

(G AP DH ) were am plified in the sam e well. In a ny par-2.1. Derivation of t he 2C T Methodt icu la r w ell, w e k n o w t h a t t h e c -m yc rea ct ion a n d t h e

G AP DH rea ct ion h a d exa ct ly t h e s a me c DN A in pu t . N orma liz in g t o a n en d ogen ou s referen ce p rov ides a

method for correcting results for differing am ounts ofTherefore, it makes sense to calculate C T separately

for each w ell. These C T va lu es ca n t h en b e a v era ged in pu t RN A. On e h a l lma rk of t h e 2C T method is that

i t u s e s d a t a g e n e r a t e d a s p a r t o f t h e r e a l - t i m e P C Rbefore proceeding with the 2C T ca lculat ion. Aga in,

t h e est ima t ed error is given a s a n a s y mmet ric ra n ge of exp erimen t t o p erform t h is n orma liz a t io n fu n ct ion .

This is part icularly at tract ive when it is not practicalva lues, reflecting conversion of a n exponent ia l var iable

t o a lin ea r com pa rison . t o m ea sure t h e a moun t of in put RNA by ot h er met h ods.

Such situa tions include when only limited a mounts ofIn Tables 1 a nd 2, the est ima ted error ha s not been

increased in proceeding from the C T column to the RNA ar e ava ilable or wh en high-throughput processing

of many samples is desired. I t is possible, though, toC T column. This is because we have decided to dis-

p la y t h e d a t a w it h error s h ow n b o t h in t h e ca l ib ra t o r n orma liz e t o s ome mea s u remen t ext ern a l t o t h e P C R

experiment. The most common method for normaliza-a nd in the test sam ple. Subtra ction of the a verage C T,cb

to determine the C T va lu e is t rea t ed a s s u b t ra ct ion t ion is t o u s e U V a b s orb a n c e t o d et ermin e t h e a mo u n t

TABLE 2

Trea tment of Replica te D a ta Where Ta rget a nd R eference a re Amplified in t he S a me Wella

c-m yc C T (Avg. c-myc C T C T (Avg. C T Normalized c-m yc a mountTissue C T G AP D H C T Avg. G APDH C T) Avg. C T,Br ain ) rela tive t o bra in 2C T

B r a in 32.38 25.07 7.3132.08 25.29 6.7932.35 25.32 7.03

32.08 25.24 6.8432.34 25.17 7.1732.13 25.29 6.84

Aver a ge 6.93 0.16 0.00 0.16 1.0 (0.9– 1.1)K idney 28.73 24.30 4.43

28.84 24.32 4.5228.51 24.31 4.2028.86 24.25 4.6128.86 24.34 4.5228.70 24.18 4.52

Aver a ge 4.47 0.14 2.47 0.14 5.5 (5.0– 6.1)

a An experiment like tha t d escribed in Ta ble 1 wa s performed except the reactions conta ined primers a nd probes for both c-m yc a ndGAPDH. The probe for c-m yc w as labeled w it h t he rep ort er dye FAM a nd t he p robe for G AP DH w a s la beled w it h t he rep ort er dye J OE .Because of the different reporter dy es, the real-time P CR signa ls for c-m yc and G AP DH can be distinguished even though both amplificat ions

are occu rring in t he sa me w ell.




of RN A a d d ed t o a cDN A rea ct ion . P C Rs a re t h en s et eq u a t ion w h ere C T C T,Tim e x C T,Time 0 (Fig. 3). Ast a -

t is t ica l ly s ign ifica n t rela t ion s h ip exist s b et w een t h eup using cDNA derived from the sa me a mount of input

RNA. One example of using this external normalizat ion trea tment an d expression of G AP DH but not for 2-

microglobulin (Fig. 3). Therefore, 2-microglobulinis to s tudy t he effect of experimental t reat ment on th e

expression of a n endogenous reference to determine if ma kes a suitable interna l control in qua ntita t ive serum

stimu lat ion studies wh ile G AP DH does not. This exa m-the int ernal control is affected by trea tment. Thus, the

t a rget gen e a n d t h e en d ogen ou s referen ce a re o n e in p le d emon s t ra t es h ow t h e 2C T met h od ma y b e u s ed

to ana lyze relat ive gene expression da ta wh en only onethe same. In this case, Eq. [2] is not divided by Eq. [3]a n d E q . [5] becom es gen e is bein g st udied.

X 0 (1 E X)C T,X K X. [10]3. STATISTICAL ANALYSIS OF REAL-TIMEPCR DATARearr a nging gives th e expression

The endpoint of rea l-time P CR a na lysis is the th resh-X 0 K X (1 E X)C T,X. [11]

old cycle or C T. The C T is determin ed from a log– linea r

plot of t he P CR signal versus the cycle number. Thus,Now, dividing X 0 fo r a n y s a mp le q b y t h e X 0 fo r t h e C T i s a n exp o n en t ia l a n d n o t a l in ea r t erm. F o r t h is

calibra tor (cb) gives rea s on , a n y s t a t ist ica l p res en t a t ion u s in g t h e ra w C T

X 0,q

X 0,cb

K X (1 E X)C T,q

K X (1 E X)C T,cb (1 E X)C T, [12]

w h ere C T i s eq u a l t o C T,q C T,cb. The prime is used

to dist inguish this expression from the previous C Tcalculat ion (see Eq. [6]) tha t involved subt ra ction of C Tvalues for target and reference.

As stated in Section 1.1, if properly optimized, the

efficiency is close to one. The amount of endogenous

reference relat ive to a ca libra tor t hen becomes

2C T. [13]

2.2. Application of t he 2C T Method

An appropriate application of the 2C T method is to

determine the effect of the experimental t reat ment on

th e expression of a candida te int erna l cont rol gene. To

demonstra te this ana lysis , serum star vat ion and induc-

tion experiment s were performed (7). Serum st a rva tion/induction is a commonly used m odel to study t he decayFIG. 3. Application of th e 2C

T meth od. The following experimentof certain mRNAs (8). However, serum may alter the

was conducted to validate the effect of treatment on the expressione xp re ss ion of n u m er ou s g en es i n cl ud in g s t a n d a r d of ca ndida te interna l control genes. NIH 3T3 fibroblast s w ere serumhousekeeping genes (9). sta rved for 24 h a nd then induced wit h 15%serum over a n 8-h period.

Sa mples were collected a t va rious t imes following serum stimula tion;Gene expression was induced in NIH 3T3 cells bymR NA w a s ext ract ed a nd convert ed t o cDNA. The cDNA w a s su b-a dding 15% serum following a 24-h period of serumjected to rea l-time q ua ntita tive P CR using gene-specific primers for

sta rva tion. P oly(A)+ RNA wa s extra cted from t he cells 2-microglobulin and GAPDH. The fold change in gene expressiona nd equiva lent a mounts w ere converted t o cDNA. The wa s calculat ed using Eq. [13], where C T (C T,Time x C T,Time 0) and

is presented for both 2-microglobulin (A) a nd G APD H (B). Reprintedamounts of 2-microglobulin an d G AP DH cDNA w erefrom T. D. S chmitt gen a nd B . A. Zakra jsek (2000)E ffect of experimen-d et ermin ed b y rea l-t ime q u a n t i t a t iv e P CR w it h S Y BRtal treatment on housekeeping gene expression: Validation by real-

G reen det ection (7). The rela tive a mounts of 2-micro- t ime qua ntita tive RT-P CR, J . Bi ochem. Biophys. Methods 46, 69– 81,

with permission of Elsevier Science.g l o b u l i n a n d G A P D H a r e p r e s e n t e d u s i n g t h e 2C T




va lues should be a voided. As described w ithin the previ- should be used. Otherw ise, presenta tion of the rela tive

gene expression should suffice. Relative quantificationous sections of th is ar ticle, present a tion of relat ive PC Rm a y b e e a s i er t o p er f or m t h a n t h e a b s ol ut e m e t h odd a t a is mo s t o f t en c a lc u la t ed a lo n g w it h a n in t ern a lbeca use the use of s tanda rd curves is not required.cont rol and /or ca libra tor sa mple an d is ra rely presented

The equa tions provided her ein should be sufficient fora s t h e C T . An exception is when one is interested inan investiga tor to ana lyze quan tita t ive gene expressionexam ining th e sa mple-to-sa mple varia tion among repli-d a t a u s in g rela t iv e q u a n t i f ica t ion . To s u mma r iz e t h ecate reactions.

imp o rt a n t s t ep s in t h e d es ign a n d ev a lu a t io n o f t h eTo d emon s t ra t e t h is , 96 rep lica t e rea ct ion s of t h eexperiment : (i ) select a n int erna l cont rol gene, (i i ) vali-identical cDNA were performed using real-t ime PCRd a t e t h e i n t e r n a l c o n t r o l t o d e t e r m i n e t h a t i t i s n o ta n d S Y B R G r e en d et e ct i on . A m a s t er m i xt u r e con -a f fec t ed b y exp erimen t a l t rea t men t , a n d (i i i ) P C R onta ining all of the ingredients w as pipett ed into individ-perform dilutions of R NA or cDNA for both the ta rgetua l tubes of a 96-w ell reaction plat e. The sa mples werea nd intern a l contr ol genes to ensure tha t th e efficienciessubjected to real-t ime P CR a nd the individual C T valuesa re similar. Fina lly, stat istical da ta should be convert edw ere d et ermin ed . To exa min e t h e in t ra s a mp le v a ria -t o t h e l in ea r fo rm b y t h e 2C T calculat ion and shouldtion, the mean SD wa s determined from th e 96 sam -not be presented by the raw C T va lues.ples. I f calcula ted from the r aw C T t h e mea n S D w a s

20.0 0.194 with a CV of 0.971%. H owever, w hen the

individual C T values w ere converted to t he linear form

REFERENCESu s in g t h e t erm 2C

T, t h e mea n S D w a s 9. 08 10

7

1.33 107 with a CV of 13.5%. As demonstra ted1. Murphy, L. D., Herzog, C. E., Rudick, J . B., Fojo, A. T., and B a tes,

b y t h is s imp le exa mp le, rep o rt in g t h e d a t a o b t a in edS. E. (1990) Biochemistry 29, 10351– 10356.

from the ra w C T values falsely represents the va riat ion 2. Noonan , K. E ., Beck, C., H olzma yer, T. A., Chin , J . E., Wunder,a nd should be avoided. Converting t he individua l dat a J . S. , Andrulis, I . L., G azda r, A. F. , Willman, C. L. , G riffith, B. ,

Von-Hoff, D . D., a nd Robinson, I. B . (1990) Proc. Natl. Acad. Sci.to a l inear form using 2C T more accurately depicts theU SA 87, 7160– 7164.

individual var iat ion a mong replica te rea ctions.3. Horikoshi, T., et al. (1992) Can cer Res. 52, 108– 116.

4. Heid, C . A., St evens, J . , Liva k, K. J . , a nd William s, P. M. (1996)Genome Res. 6, 986– 994.

5. Winer, J . , J ung, C. K., S ha ckel, I . , an d Willia ms, P. M. (1999)4. CONCLUDING REMARKSAnal . Bi ochem. 270, 41– 9.

6. Schm itt gen, T. D., Zakra jsek, B . A., Mills, A. G., G orn, V., Singer,

E x p e r i m e n t a l d e s i g n a n d d a t a a n a l y s i s f r o m r e a l - M. J ., a nd Reed, M. W. (2000) Anal . Bi ochem. 285, 194– 204.t ime, q u a n t i t a t iv e P CR exp erimen t s ma y b e a c h iev ed 7. Schmittg en, T. D., an d Za krajsek, B. A. (2000) J. Bi ochem. B io-

phys. Methods 46, 69– 81.using either relat ive or a bsolute q uan tifica t ion. When8. Ch en, C. Y., and S hyu, A. B. (1994) M ol. Cell . Biol. 14,8471– 8482.designing quantitat ive gene expression studies using9. Iyer, V. R., et al. (1999) Science 283, 83– 87.

rea l-t ime P CR, t h e f irst q u est ion t h a t a n in v es t iga t o r10. G iulietti, A., Overbergh, L. , Va lckx, D., D ecallone, B . , B ouillon,

should ask is how should the da ta be presented. If a bso- R. , a nd Ma thieu, C. (2001) M ethods 25, 386– 401.

11. Niesters, H. G . M. (2001) M ethods 25, 419– 429.lute copy number is requ ired, then t he absolute meth od

livak_schmittgen2001

Documents