How do you select 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 are reference genes used 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 much genomic DNA do you need for qPCR?
What is the minimum amount of genomic DNA required for analysis using EpiTect Methyl qPCR Arrays? For digestion, we recommend using 0.5-4ug (0.125-1ug per digestion) of genomic DNA for EpiTect Methyl qPCR Arrays and 0.25-0.5ug (0.0625-0.125ug per digestion) for single qPCR assays.
How many housekeeping genes are needed for RT-PCR?
13 housekeeping genes
We used real-time reverse transcription PCR (RT-PCR) to assess the levels of 13 housekeeping genes expressed in peripheral blood mononuclear cell culture and whole blood from healthy individuals and those with tuberculosis.
How do you choose a reference gene?
The ideal reference gene A mRNA used as reference or standard of a QRT-PCR (and other experiments) should have the following properties: expressed in all cells. constant copy number in all cells. medium copy number for more accuracy (or similar copy number to gene of interest)
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 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.
How much DNA do you need for real time PCR?
Real-time PCR can measure the initial concentration of target DNA over a range of 5 or 6 orders of magnitude. At present, the limit of detection when fluorescent dyes are used is ≈10–100 copies of template DNA in the starting reaction (Sambrook and Russell 2001).
Can you use genomic DNA for qPCR?
As genomic DNA contains both exons and introns you cannot look at the expression of a particular gene. However you can still use your genomic DNA in qPCR which wouldn’t give you the exact expression of the gene you are looking for.