How do you select reference genes for qPCR?

The best way of selecting the most appropriate control gene for a relative qPCR experiment is to select some candidate genes and determine their expression levels across the range of experimental conditions and treatments. The genes most stably expressed across these conditions will be the most appropriate controls.

What is reference gene in qPCR?

The identification of gold-standard reference genes is crucial to produce reliable qPCR results. The expression of reference genes is used to correct the fluctuations in the target gene expression levels caused by technical variations in the quantity of total RNA or in the cDNA synthesis.

How many reference genes are needed for qPCR?

Finally, the MIQE guidelines suggest the use of at least two reference genes and to test whether more than two are necessary42. Considering our results, in all the three models tested in our study, at least two reference genes are enough for a strong and reliable normalisation in RT-qPCR experiments.

What are good housekeeping genes for qPCR?

Most stable genes were Atp5f1, Pgk1 and Gapdh, while the least stable genes were Rps18, Hprt, Tbp and Actb. The results were validated by a proof-of-point qPCR experiment with pluripotent markers Nanog, Rex1 and Oct4 normalized to the best and the worst reference gene identified by the analyses.

How do you Analyse qPCR results with multiple reference genes?

How to use the equation

1. Calculate primer efficiencies.
2. Select a calibrator sample to determine delta Ct (∆Ct)
3. Calculate delta Ct (∆Ct) values.
4. Calculate relative quantity (RQ) values.
5. Calculate the geometric mean of the reference genes RQ values.
6. Calculate relative gene expression values.

What are the main criteria for selecting a reference gene?

A reference gene is a gene whose expression is stable in all studied conditions. It would ideally have exactly the same expression level in all samples, with only measurement noise added.

What is a good reference gene?

For a gene to be regarded as a reliable reference it must meet several important criteria (Chervoneva et al. 2010). The most important is its expression level unaffected by experimental factors. Also, it should show minimal variability in its expression between tissues and physiological states of the organism.

What is the difference between PCR and qPCR?

The conventional PCR can only amplify the DNA up to 2000 nucleotides precisely while the rtPCR or qPCR can amplify DNA as well as quantify the amount of DNA as well. Quantification of nucleic acid measures how much DNA templates are present in the sample.

How does qPCR measure gene expression?

If you are measuring gene expression, qPCR will tell you how much of a specific mRNA there is in your samples. You amplify a small region of this mRNA with oligos and a fluorescent probe (if working with Taqman). The qPCR machine measures the intensity of fluorescence emitted by the probe at each cycle.

Is GAPDH a good housekeeping gene?

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a well-known housekeeping gene with functions in glycolysis. Many biologists are familiar with the gene and use GAPDH antibodies for a loading control when performing western blots.

How are reference genes used in quantitative RT-PCR?

For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting.

Which is the best model for gene expression profiling?

In animal-based toxicological studies, the rat has become a tractable model and gene expression profiling is often performed. Studies also often include an array of different tissues, perhaps from various stages of development.

Which is the best method to select reference genes?

This approach using the comparative Ct (or 2−ΔCt) method (Schmittgen&Livak,2008) is sound, but rests on the assumption that transcript abundance of the reference gene is the same in all cells, under all conditions.