How do I choose a reference gene 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 a 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?
two reference genes
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 is a good reference gene for qRT-PCR?
Seven common reference genes, such as 18S, 28S, ACT, GAPDH, EF1α, RPL7 and TBP, were selected and evaluated for target gene expression normalization using the qRT-PCR method in BGA. Our results revealed that specific reference genes were required for qRT-PCR normalization under various experimental conditions.
How do you choose a housekeeping gene for PCR?
The prerequisite of a suitable housekeeping gene is that it should, of course, be adequately expressed in the tissue of interest, but most importantly, that it shows minimal variability in expression between samples and under the experimental conditions used [2, 4].
What is housekeeping gene in qPCR?
Housekeeping genes are cellular maintenance genes which regulate basic and ubiquitous cellular functions. In many RT-qPCR reactions, these genes are used as internal control genes without proper validation.
What are reference genes used for?
Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances.
What is the purpose of a reference gene?
Reference genes are an internal reaction control that have sequences different than the target. 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.
How does RT-qPCR measure gene expression?
RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a …
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 are good housekeeping genes for qPCR?
Considering all the passages analysed the best housekeeping gene to normalize a RT-qPCR study on human pancreatic organoids were RPL13A (geo. mean = 1.19), and HPRT (geo. mean = 1.73) whereas the least stable HKG were RNA18S (geo.
How many housekeeping genes are needed for qPCR?
Housekeeping genes encode proteins that are usually essential for the maintenance of cellular function and often remains constant under most experimental conditions. With this array, you can easily profile the expression of twelve (12) commonly used housekeeping genes in eight (8) samples using SYBR-green based qPCR.