Primer validation/efficiency by qRT-PCR in five simple steps

You have now amplified and measured the Ct of your serial cDNA dilutions... Now what?😅

To measure the efficiency of your primer couple, you have probably been recommended the serial dilution method💧🌡️

This method involves making dilutions of your typical control sample (e.g., 1 in 4, 1 in 16, 1 in 64, 1 in 256, 1 in 1024) and mixing it with your typical Master mix and your favorite primer concentration.

This way you can confirm in silico whether the amplification pattern of your primers in successively diluted samples fits a trend line.

How to measure the efficiency of your primer couple using the serial dilution method💦

1) If you measured Ct values ​​from at least 3 replicates of each dilution, calculate the Ct average of the 3 replicates for each dilution.

2) Consider your 1 in 4 sample as a hypothetical cDNA concentration value of 100 (100%), since that will be the highest sample concentration point. The remainder will be: 1 in 16 (25%), 1 in 64 (6.25%), 1 in 256 (1.5625%), 1 in 1024 (0.3906%).

3) Apply the decimal logarithm to the percentages. So: log 100, log 25, log 6.25 and log 1.5625.

4) Plot your 5 points and draw the trend line. To do this, keep in mind that on the Y axis you must represent the average Ct values ​​for each dilution (mean of the triplicate); and on the X axis the logarithm of your hypothetical cDNA concentration.

5) Enter the slope value of your trend line into the primer efficiency formula for qRT-PCR: 

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

Here is an example:

How do I know if my efficiency value is good or bad?👮

👉If you substitute the slope into the formula, you should get an efficiency value between 80-120. A value of 100 is optimal (your primer pair is working perfectly!).👊

Didn't it go well?😓

👉Please note that the most scientifically accepted value is for Ct ​​to be less than 30. However, Ct values ​​equal to or slightly greater than 30 could be observed in your more dilute samples.

If this compromises the efficiency of your primers, consider using fewer points on your line (use less than 5 cDNA dilutions), especially if the Ct values ​​of your more diluted samples are higher than those of your water and non-reverse transcribed RNA (a.k.a. -RT) negative controls.

👉These last two controls should show INDETERMINATE Ct values ​​or, in any case, very high (Ct>30 or 40), indicating a minimal DNA contamination in the water and/or the -RT sample. And if it always occurs it could be due to contamination in the reagents of your Master mix or in the stock dilution of your primer pair (sorry..., you will have to prepare them again😥).

Some considerations👽

👉If the slope of your efficiency line is not around a value of about -3, then one of the problems we discussed above is probably occurring.

👉The ordinate at the origin must be between 25-30, otherwise, suspect one of the problems in the previous section (Didn't it go well?).

Please note that if you follow this protocol, serial dilutions should be made from a stock concentration of approximately 100 ng/ml reverse transcribed DNA (cDNA).

J.