8/19/2019 Experimental Setup and Controls for QPCR

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Experimental setup and controls for qPCR 

For accurate analysis of qPCR results, each experiment needs to be set up with multiple replicates

and controls (Figure 2)

 Replicates: For each experimental and control sample to be compared, at least three technical

replicates are necessary to minimi!e errors in measured gene expression due to pipetting

Figure 2: qPCR assay setup. "utline

of the test and controls needed for an

experiment with two different samples

examined at se#eral time points post

treatment $nclude %&' biologicalreplicates for each time point studied

For each biological replicate studied,

 perform 2 re#erse transcription

reactions (R) and * with no R (& 

R control) For each c+- sample

generated, set up % technical replicates

for qPCR analysis $nclude a o

emplate Control for each gene

analy!ed to identify any signal due to

contamination

Controls

 No RT control: For e#ery re#erse

transcription reaction, it is important to

incorporate a .no R control/ to identify erroneous signal due to genomic +- contamination

 No template control: +uring qPCR setup, incorporate a .no template control/ for e#ery different

gene analy!ed to identify possible cross0contamination generated during preparation of samples

 Reference genes: 1ultiple reference genes should be analy!ed for stable gene expression elect a

reference gene with an expression pattern that is unaltered by treatment or the different time points to

 be tested

Cycling conditions: -lways follow the cycling conditions according to the master mix used For example, do not use a standard master mix with fast cycling conditions