11/12/2023 0 Comments Deltagraph 6![]() ![]() However, this is difficult when the two experimental groups vary in n numbers and do not have matched pairs.Īnother way to select a calibrator/reference sample is to pick the sample with the highest Ct value, so the sample with the lowest gene expression. This is all well and true for experiments that have matched pairs, such as the case in cell culture experiments. Basically, this all depends on your experiment set-up.Ī common way of doing this is to just match the experimental samples and determine the relative gene expression ratios separately. This is the part which confuses a lot of people. The next step is to decide which sample, or group of samples, to use as a calibrator/reference when calculating the delta-delta Ct ( ∆∆Ct) values for all the samples. Select a calibrator/reference sample(s) to calculate delta delta Ct The formula to calculate delta Ct is presented below.įor example, to calculate the ∆Ct for the ‘ Control 1‘ sample:ģ. ![]() The next step is to calculate delta Ct ( ∆Ct) for each sample by using the newly created average Ct values. In the example below, each sample was run in duplicate ( Ct1 and Ct2). So, when performing the qPCR in duplicate or triplicate, for example, these values need to be averaged first. The first step is to average the Ct values for the technical replicates of each sample. Average the Ct values for any technical replicates Here is how to calculate the relative gene expression in 5 easy steps. If you have more than one housekeeping gene, it may be worth checking out the guide on analysing qPCR data with numerous reference genes. To use the delta-delta Ct method, you require Ct values for your gene of interest and your housekeeping gene for both the treated and untreated samples. Using the delta-delta Ct formula to calculate gene expression This is to essentially normalise the gene of interest to a gene which is not affected by your experiment, hence the housekeeping gene-term. ∆Ct = Ct (gene of interest) – Ct (housekeeping gene)īasically, ∆Ct is the difference in Ct values for your gene of interest and your housekeeping gene for a given sample. ∆∆Ct = ∆Ct (treated sample) – ∆Ct (untreated sample)Įssentially, ∆∆Ct is the difference between the ∆Ct values of the treated/experimental sample and the untreated/control sample. So, let’s take a look to see what the ∆∆Ct part of the equation means: So it is useful to use when summarising long formulas. Delta is a mathematical term used to describe the difference between two numbers. Simply, it is the cycle number where the fluorescence generated by the PCR produce is distinguishable from the background noise. This is given after the qPCR reaction by the qPCR machine. Let’s break the formula down into easier to understand chunks.įirstly, Ct stands for the cycle threshold (Ct) of your sample. This looks like a scary mathematical formula when in actual fact, it isn’t. The overall formula to calculate the relative fold gene expression level can be presented as: It is worthwhile understanding what the delta-delta Ct formula means before diving straight into the calculations. Understanding the delta-delta Ct method formula If you would like to download this, simply click here. Everything is done for you, all that is required is the Ct values! Use this to practise and get the hang of the calculations. We have created a FREE Excel template which contains all of the formula described in this article below. >Use code 20QPCR to get 20% off<< The FREE Microsoft Excel template Mastering qPCRįurther video tutorials on qPCR data analysis can be found in our Mastering qPCR course The method was devised by Kenneth Livak and Thomas Schmittgen in 2001 and has been cited over 61,000 times. The delta-delta Ct method, also known as the 2 – ∆∆Ct method, is a simple formula used in order to calculate the relative fold gene expression of samples when performing real-time polymerase chain reaction (also known as qPCR). ![]()
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