Real-Time PCR (Quantitative PCR)

Goals

1. Understand the fundamental difference between qPCR and traditional PCR

2. Understand the basic quantification method using of qPCR

3. Understand differences between qPCR and Northern blotting

Applications of real-time PCR

• Powerful and reliable quantitative method– Gene expression– Determination/monitoring of viral load– Quantification of cancer genes– Microarray verification– Transgenic copy– SNP analysis

• Three phases

Linear phase

Plateau phase

Exponential phase

PCR cycle number

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Steps of real-time PCR

Xn = X0 * (1 + E) n

E = [10(–1/slope)] – 1(Efficiency = 1 during exponential amplification)

Exponential amplification of PCR

Xn = DNA copies at cycle nX0 = DNA copies at cycle 0E = efficiency of amplificationn = cycle number

• Fluorescence detection system

• Two types of fluorochromes– DNA binding dye– Probe-based fluorochromes

Quantitative detection system

SYBR green (DNA binding dye)

Most commonly used

SYBR green

Probe-based fluorochromes (FAM, VIC, TET, FRET)

Less commonly used now

Fluorophore Quencher

• Does not discriminate between the gene of interest and other DNAs (i.e. contamination)

• Does not allow to do multiplex PCR

• Requires less steps • Less costly

• Does discriminate, more specific

• Allows multiplex PCR with usage of different fluoro.

• Requires multiple steps• More costly

SYBR green Probe-based Fluoro.Vs.

PCR cycle number

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Detection zones qPCR vs PCR

Traditional PCR with EtBr

qPCR

• Fluorescence increase is proportional to DNA amplification

• The first cycle at which the instrument can distinguish the amplified fluorescence as being above the background level is called the threshold cycle or “Ct”

Amplicon quantification by qPCR

The threshold cycle (Ct)

Ct

Example of a Ct curve

The threshold cycle (Ct)

Ct curves of three different samples.

• The Ct value is inversely proportional to the starting concentration of the sample

– i.e. the greater the amount of DNA in the sample the lower the Ct value

The threshold cycle (Ct)

1. Absolute quantification – To determine exact amounts of DNA (e.g. viral load)

2. Relative quantification– To determine changes in gene expression

Quantification methods

• If initial amount of DNA copies is known:

XT = X0 * (1 + E) Ct

• If not, Ct values of the samples has to be compared to a standard curve

Absolute quantification

XT = DNA copies at thresholdX0 = DNA copies at cycle 0E = efficiency of amplificationCt = threshold cycle

-1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 015.00

17.00

19.00

21.00

23.00

25.00

f(x) = − 3.13922204966855 x + 18.221R² = 0.999253657859755

Standard Curve

Log of DNA concentration

Cycle

s

Absolute quantification

Sample of Mel1 gene which had a Ct of 22.5 cycles after amplification. What is the concentration of your amplicon?

Absolute quantification

22.5 = -3.1392 x + 18.221

Concentration of Mel1 amplicon with Ct of 22.5

x = -1.3630

The DNA concentration is 0.043 µg/ml

10 -1.3630 (inverse Log 10)

y = -3.1392 x + 18.221

• Normalization of the gene of interest to a housekeeping gene

Relative quantification

Sample

Housekeeping¿Ratio

• More sensitive (need ~50 ng)

• More accurate (can determine numbers)

• DNA template (stable)• Doesn’t give size of

transcripts• Faster (few hours)• Requires less steps• Less costly

• Less sensitive (need ~10 ug)

• Less accurate (cannot determine copy numbers)

• RNA template (unstable)• Gives size of transcripts• Long (hours to days)• Requires numerous

processing steps• More costly

Real-time PCR Northern blottingVs.