PCR quantitativo

What is Real-Time

PCR?

Real-Time PCR is a specialized technique that allows a PCR reaction to be visualized “in real time” as the reaction progresses.

This enables researchers to quantify the amount of DNA in the sample at the start of the reaction!

What is Real-Time

PCR? • 20ul PCR reactions• SYBR Green or probes

94°C 4 min 94°C 15 sec 61°C 30 sec 72°C 30 sec

40x

Differences with normal PCR?

What is Real-Time PCR used

for?

Real-Time PCR has become a cornerstone of molecular biology:

• Gene expression analysis– Medical research– Drug research

• Disease diagnosis– Viral quantification

• Food testing– Percent GMO food

• Transgenic research– Gene copy number

Taq polymerase can only synthesize DNA, so how do we study gene expression (RNA) using qPCR?

What is Real-Time

PCR?

Reverse transcriptionRNA -> DNA (cDNA)

What’s Wrong With Agarose Gels?

Low sensitivity Low resolution Non-automated Size-based discrimination only Results are not expressed as numbers based on personal evaluation Ethidium bromide staining is not very

quantitative End point analysis

Different concentrations give similar endpoint results!

Endpoint analysis

Imagining Real-Time

PCR

…So that’s how PCR is usually presented.

To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…

Imagining Real-Time

PCR What’s in our tube, at cycle number 25?

A soup of nucleotides, primers, template, amplicons, enzyme, etc.

~1,000,000 copies of the amplicon right now.

Imagining Real-Time

PCR

How did we get here?

What was it like last cycle, 24?

Almost exactly the same, except there were only 500,000 copies of the amplicon.

And the cycle before that, 23?

Almost the same, but only 250,000 copies of the amplicon.

And what about cycle 22?

Not a whole lot different. 125,000 copies of the amplicon.

Imagining Real-Time

PCR

How did we get here?

If we were to graph the amount of DNA in our tube, from the start until right now, at cycle 25, the graph would look like this:

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?

Imagining Real-Time

PCR

So where are we going?

What’s it going to be like after the next cycle, in cycle 26?Probably there will be 2,000,000 amplicons.

And cycle 27?Maybe 4,000,000 amplicons.

And at cycle 200?In theory, there would be

1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…

Imagining Real-Time

PCR

So where are we going?

If we plot the amount of DNA in our tube going forward from cycle 25, we see that it actually looks like this:

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• Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out.

• So exponential growth does not go on forever!

Imagining Real-Time

PCR

MeasuringQuantities

How can all this be used to measure DNA quantities??

What if YOU started with FOUR times as much DNA template as I did?

I have 1,000,000 copies at cycle 25.

You have 4,000,000 copies!

So… You had 2,000,000 copies at cycle 24.

And… You had 1,000,000 copies at cycle 23.

Imagining Real-Time

PCR

MeasuringQuantities

So… if YOU started with FOUR times as much DNA template as I did…

Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!

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Imagining Real-Time

PCR

MeasuringQuantities

What if YOU started with EIGHT times LESS DNA template than I did?

You’d only have 125,000 copies right now at cycle 25…

…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle 28 you’ll have caught up with 1,000,000 copies!

So… you’d reach 1,000,000 copies exactly THREE cycles later than I would!

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Imagining Real-Time

PCR

MeasuringQuantities

• The value that represents the cycle number where the amplification curve crosses an arbitrary threshold.

• Ct values are directly related to the starting quantity of DNA, by way of the formula:

Quantity = 2^Ct

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Ct Values:

Threshold

The “ct value”

threshold

Ct

Imagining Real-Time

PCR

MeasuringQuantities

There’s a DIRECT relationship between the starting amount of DNA, and the cycle number that you’ll reach an arbitrary number of DNA copies (Ct value).

DNA amount = 2 ^ Cycle Number

Copy Number vs. Ct - Standard Curve

y = -3.3192x + 39.772

R2 = 0.9967

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Log of copy number (10n)

Ct

How do We

Measure DNA in a

PCR Reaction?

We use reagents that fluoresce in the presence of amplified DNA!

Ex. SYBR Green dye

They bind to double-stranded DNA and emit light when illuminated with a specific wavelength.

How do We

Measure DNA in a

PCR Reaction?

Extension

5’ 3’

5’3’5’ 3’

5’3’

Apply ExcitationWavelength

5’ 3’

5’3’5’

5’

Taq

Taq

3’

5’3’

Taq

Taq5’

5’

ID ID

ID IDID

ID ID ID

ID ID

l l l

ll

SYBRgreen • dsDNA intercalating dye• Unspecific (optimization)• cheap

Melting curve – Test the presence of unspecific amplification, contamination, primer dimers,..

TaqMan probes • Sequence-specific• Doesn´t need much optimization• More expensive

What Type of

Instruments are used

with Real-Time PCR?

Real-time PCR instruments consist of TWO main components:• Thermal Cycler (PCR machine)• Optical Module (to detect

fluorescence in the tubes during the run)

What Type of

Instruments are used

with Real-Time PCR?

• Adequate, optical plates 96/384 wells Standard/fast

• Optical sealing adhesive

Quantificationand

Normalization

Quantification and Normalization

• First basic underlying principle: every cycle there is a doubling of product.

• Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities.

• Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene.

• Key formula:

Quantity = 2 ^ (Cta

– Ctb

)

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Quantification and Normalization

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Standard Curve

Prepare a 2-fold serial dilution of a DNA sample:

Recomendation: add always a standard curve in every run

“normalizer” geneQuantification and

Normalization

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• Knowing the amount of mRNA in one sample from one specific gene does not tell us much..

• You need to know the total amount of mRNA in your sample

• You also dont know how much the mRNA level has changed compared to other mRNA levels

• Example:mRNA levels of a gene increase 2x after induction

It is possable that all (1) genexpression in the cell has increased (2) the induced samples contained more total mRNA

We have to compare the expression of our gene to another gene which expression is normally constant, a housekeeping gene (ex. TBP, 18S)

ΔΔCt method

2-[(Cttg-Ctcg)-(Cttg-Ctcg)]

experiment control

Always in duplicate or triplicate!

DCt = target gene– ref geneD Ct = 9.70

Difference = DCt-DCt= DDCt = 9.70-(-1.7)= 11.40Fold change = 211.40 = 2702

DCt = target gene– ref geneD Ct = -1.70

Ex!

• Sempre testar os primers pela 1ª vez por PCR normal em pelo menos 3 diferentes temperaturas e correr gel!

Escolher a máxima temperatura em que há apenas 1 banda (e do tamanho esperado!) e a amplificação ainda é satisfatoria!

• Para qPCR: Iniciadores que flanqueiam a junção exon-exon para evitar amplificação inespecífica devido à contaminação por gDNA.

Quanto aos primers..

Calculando a eficiência dos primers em qPCR

Curva padrão

threshold

Ct

Ideal: N = N0.2n

N = número de moléculas amplificadasN0 = número de moléculas inicialn = Número de ciclos

15SERIES OF 10-FOLD DILUTIONS

threshold

35

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PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A100% EFF

90% EFF

80% EFF

70% EFF

CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY

0 1 1 1 11 2 2 2 22 4 4 3 33 8 7 6 54 16 13 10 85 32 25 19 146 64 47 34 247 128 89 61 418 256 170 110 709 512 323 198 11910 1,024 613 357 20211 2,048 1,165 643 34312 4,096 2,213 1,157 58313 8,192 4,205 2,082 99014 16,384 7,990 3,748 1,68415 32,768 15,181 6,747 2,86216 65,536 28,844 12,144 4,86617 131,072 54,804 21,859 8,27218 262,144 104,127 39,346 14,06319 524,288 197,842 70,824 23,90720 1,048,576 375,900 127,482 40,64221 2,097,152 714,209 229,468 69,09222 4,194,304 1,356,998 413,043 117,45623 8,388,608 2,578,296 743,477 199,67624 16,777,216 4,898,763 1,338,259 339,44925 33,554,432 9,307,650 2,408,866 577,06326 67,108,864 17,684,534 4,335,959 981,00727 134,217,728 33,600,615 7,804,726 1,667,71128 268,435,456 63,841,168 14,048,506 2,835,10929 536,870,912 121,298,220 25,287,311 4,819,68630 1,073,741,824 230,466,618 45,517,160 8,193,466

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PCR CYCLE NUMBER

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A

100% EFF

90% EFF

80% EFF

70% EFF

E = 10^(-1/slope)E = (10^(-1/slope)-1)*100

Ex. slope = -3,81

Eficiência = (10^(-1/-3.81)-1)*100 = 83%

Ideal: eficiência >95%

Gene Value CallA 1.828745739 up regulatedB 2.04179718 up regulatedC 0.666738198 unaffectedD 1.999855536 up regulatedE - 0.450673805 unaffectedF 0.509327854 unaffectedG - 1.195371388 down regulatedUCE 0 control gene

-1.5

-1

-0.5

0

0.5

1

1.5

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2.5

Relative gene expression

Treatment

log1

0 of

rela

tive

gene

exp

resio

n

Results?

Adequate nr of samples Adequate nr of replicates

Good STATISTICS

Good experimental designOptimal primers

Good RNAGood cDNA