What is a good primer

• It is a short sequence, 16-24bp

• It binds spontaneously to the target

• It does not bind easily to other things

• It works well with the other primer

• It works well with the polymerase

In other words

• It has to be thermodynamically stable

• It has to be taxonomically specific

These two conditions imply that the sequence must be short, but not too short

Stability

• should not form hairpins

• should not form homodimers

• should not form heterodimers

• should be stable in the 3’ end

  • so the polymerase can extend

Specificity

• should match the target organism only once

  • even if there are some mismatches

• should not match other organisms

  • even if there are some mismatches

Four possible outcomes

When we use a primer to detect a target, 4 things can happen.
The primer either

• binds to the target (True Positive)

• binds to something else (False Positive)

• does not bind to the target, even if the target is there (False Negative)

• does not bind to anything, and the target was not there (True Negative)

Evaluation

When we test a possible primer, the four outcomes may happen

We measure the number of each case, using a database of all possible sequences

• \(TP\): True Positive number

• \(TN\): True Negative number

• \(FP\): False Positive number

• \(FN\): False negative number

Sensitivity and Specificity

\[ \begin{aligned} \text{Sensitivity}&=\frac{TP}{TP+FN}=\frac{\text{Detected}}{\text{All targets}}\\ \text{Specificity}&=\frac{TN}{TN+FP}=\frac{\text{Not targets}}{\text{Not detected}} \end{aligned} \]

“Say the truth, all the truth, nothing but the truth”

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is a method used to synthesize millions of copies of a given DNA sequence.

A typical PCR reaction consists of series of cycles:

• template DNA denaturation,
• primer annealing, and
• extension of the annealed primers by DNA polymerase.

This loop is repeated between 25 and 30 times

qPCR can answer several questions

• Presence of a specific mRNA molecule in the sample
• Concentration of that mRNA in the sample
• Change in concentration of that mRNA between two samples

(you can replace “mRNA” for other keywords)

How to bake your cake

Use the following ingredients

• Template: the thing we want to study
• Primers for that template, in high concentration
• dNTP, in high concentration
• Taq polymerase
• Some marker, such as a fluorophore
• Salt & pepper (Na++, K+, Mg++)
• Cooking machine: thermocycler with sensors

The thermocycler is a computer.
You program it to cook your cake

Signal is a function of concentration

We will call \(X(C)\) to the signal measured when DNA concentration is \(C\)

We are mainly interested in the initial concentration \(X(0)\)

Finding the initial concentration

We care only about the exponential phase

The signal increases 2 times on every cycle

\[X(C) = X(0)⋅2^C\]

So we can find the initial concentration

\[X(0) = X(C)⋅2^{-C}\]

CT: cycle where Signal crosses threshold

DNA concentration crosses 50% at 13.73 cycles

Standard curve gives concentration

If everything is right, we get

\[X(0) = X(CT)⋅2^{-CT}\]

But sometimes we do not know \(X(CT)\)

because the Signal is not 100% DNA concentration

It depends on the fluorophore assimilation

Delta CT

We still can measure change of concentration

Let’s say we extract mRNA before and after a shock

\[ \begin{aligned} X_B(0) & = X_B(CT_B)⋅2^{-CT_B}\\ X_A(0) &= X_A(CT_A)⋅2^{-CT_A}\end{aligned} \]

therefore the fold change of expression is

\[\frac{X_B(0)}{X_A(0)} = \frac{X_B(CT_B)⋅2^{-CT_B}}{X_A(CT_A)⋅2^{-CT_A}}\]

Fold change of expression

If we assume that the DNA concentrations are the same when the signal crosses the threshold, i.e.

\[X_B(CT_B)=X_A(CT_A)\] then \[\frac{X_B(0)}{X_A(0)} = 2^{-(CT_B-CT_A)} = 2^{-ΔCT}\]

\(Δ CT\) is the change in CT for one gene between two conditions

Why does the CT change?

This change of concentration has two components

• The real biological change
• The variability of the RNA extraction protocol

To avoid the second component, we use an endogenous reference

(typically, a housekeeping gene)

Delta Delta CT

We normalize each sample

\[ \begin{aligned} \frac{X_B(0)}{R_B(0)} &= \frac{X_B(CT_{XB})⋅2^{-CT_{XB}}}{R_B(CT_{RB})⋅2^{-CT_{RB}}}= K_B⋅ 2^{-(CT_{XB}-CT_{RB})}\\ \frac{X_A(0)}{R_A(0)} &= \frac{X_A(CT_{XA})⋅2^{-CT_{XA}}}{R_A(CT_{RA})⋅2^{-CT_{RA}}}= K_A⋅ 2^{-(CT_{XA}-CT_{RA})} \end{aligned} \]

\(K_A\) and \(K_B\) are constants that depend on the target and reference genes, and how each Signal changes with concentration

Ratio of relative expressions

\[ \frac{X_B(0)}{R_B(0)}÷\frac{X_A(0)}{R_A(0)} = \frac{K_B}{K_A} ⋅ 2^{-(\Delta CT_B-Δ CT_A)} \]

We can assume that \(K_B=K_A,\) because we are comparing the same pair of genes every time

In that case the change in relative expression is

\[\frac{X_B(0)}{R_B(0)}÷\frac{X_A(0)}{R_A(0)} = 2^{-(\Delta CT_B-Δ CT_A)}\]

Log fold change

It is usually a good idea to take logarithms

Using \(\log_2\) we get log fold change, which can be written as

\[ \begin{aligned} Δ CT_A - Δ CT_B &= \overbrace{(CT_{BX}-CT_{BR})}^{\text{before}} - \overbrace{(CT_{AX}-CT_{AR})}^{\text{after}}\\ &=\underbrace{(CT_{BX}-CT_{AX})}_{\text{target}} - \underbrace{(CT_{BR}-CT_{AR})}_{\text{reference}} \end{aligned} \]

This works very well with a linear model

We can change the order of deltas

The reordering of deltas is equivalent to

\[ \frac{X_B(0)}{R_B(0)}÷\frac{X_A(0)}{R_A(0)} = \frac{X_B(0)}{R_B(0)}⋅ \frac{R_A(0)}{X_A(0)} = \frac{X_B(0)}{X_A(0)}÷\frac{R_B(0)}{R_A(0)} \]

In other words, the ratio of normalized values is also the ratio between ratios of change

(I think the formula is more clear than the text)

Corrected formula

If the primer efficiency is \(E_X,\) the correct formula is

\[ \begin{aligned} \frac{X_B(0)}{X_A(0)} &= \frac{X_B(CT_{BX})⋅(1+E_X)^{-CT_{BX}}}{X_A(CT_{AX})⋅(1+E_X)^{-CT_{AX}}}\\ &=(1+E_X)^{-(CT_{BX}-CT_{AX})} \end{aligned} \]

The efficiencies may be different for the housekeeping gene

\[ \begin{aligned} \frac{R_B(0)}{R_A(0)} &= \frac{R_B(CT_{BR})⋅(1+E_R)^{-CT_{BR}}}{R_A(CT_{AR})⋅(1+E_R)^{-CT_{AR}}}\\ &=(1+E_R)^{-(CT_{BR}-CT_{AR})} \end{aligned} \]

If any efficiency is not 100%

\[ \frac{X_B(0)}{X_A(0)}÷\frac{R_B(0)}{R_A(0)}= \frac{(1+E_X)^{-(CT_{BX}-CT_{AX})}}{(1+E_R)^{-(CT_{BR}-CT_{AR})}} \]

The log ratio is \[-F_X(CT_{BX}-CT_{AX})+ F_R(CT_{BR}-CT_{AR})\] where \(F_X=-\log_2 (1+E_X)\) and \(F_R=-\log_2 (1+E_R)\) are the slopes of the corresponding standard curves

Calculating the efficiencies

We know the dilutions, the CT values, and their relationship \[X(0) = X(C)⋅(1+E)^{-C}\] We can connect several initial concentrations with their \(CT\)

\[ \text{initial}_i = \text{threshold}⋅(1+E)^{-CT_i} \]

Notice that threshold concentration should be the same for all \(i\)

Taking logarithms

Now we have

\[ \log(\text{initial}_i) = \log(\text{threshold}) + \log(1+E)⋅ -CT_i \]

This is an equation of a straight line, like \[y=a+b\,x\]

We care about the slope coefficient

We can find \(E\) using a linear model like \[\log_2(\text{initial}_i) = β_0 + β_1 ⋅ CT_i + e_i\]

Fitting the linear models gives us \[β_1 = -log(1+E)\] and from there we can find the primers efficiency \(E\)

Second set of primers

For the second set of primers, we have

             2.5 % 97.5 %
(Intercept)  0.108  0.566
CT          -0.606 -0.582

The efficiency is in the interval

 2.5 % 97.5 %
 0.833  0.790

The real value is 80%

It is recommended to always do the standard curve by triplicate

Threshold in the machine is not perfect

All our measuring devices have a margin of error.

There may be a small error measuring the signal.

That will affect the resulting CT value

Lower thresholds are more sensitive

When the initial concentration is small, the signal may not reach 50% in 30 or 40 cycles

Then we will use a lower threshold, and pay the price

Margin of Error

Since the curve is nearly flat in the lower signals, a small error in the signal has a large impact in the concentration

An error between signal 0.05 and 0.06 results in an apparent increase of 16% in concentration. An error between signal 0.5 and 0.51 results in an error of 3%

References

Livak KJ, Schmittgen TD. “Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method”. Methods. 2001 Dec; 25(4):402-8. doi: 10.1006/meth.2001.1262

Pfaffl, Michael W. “Relative Quantification.” In Real-Time PCR, Published by International University Line (Editor: T. Dorak), 64–82, 2007.