Why is GAPDH used in PCR?
As one of the most common reference genes, GAPDH is often used to normalize the gene expression data, being used as an endogenous control in the quantitative analysis of RT-PCR, since in some experimental systems, its expression is very constant (Edwards and Denhardt 1985; Winer et al. 1999).
Is GAPDH a primer?
Primers specific to the gene GAPDH, or glyceraldehyde 3-phosphate dehydrogenase, are often used in PCR-based experiments as a type of control. GAPDH is an enzyme involved in glycolysis and is widely expressed among many mammalian cell types.
Can PCR primers be used for RT-PCR?
One-step RT-PCR is always performed with gene-specific primers as the downstream PCR primer is also the primer for reverse transcription. In two-step RT-PCR, the three types of primers, or combinations thereof, can be used for reverse transcription.
How do I choose primers for RT-PCR?
PCR primer design IDT recommends that you aim for PCR primers between 18 and 30 bases; however, the most important considerations for primer design should be their Tm value and specificity. Primers should also be free of strong secondary structures and self-complementarity.
What is GAPDH?
What is GAPDH? GAPDH, a multifunctional protein, not only catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate during glycolysis, but also regulates multiple cellular functions including membrane fusion, transport, apoptosis, DNA replication and repair, and regulation of transcription and translation.
Is GAPDH a good housekeeping gene?
1.12) is an enzyme of ~37 kDa that plays an important role in glycolysis. GAPDH is a popular housekeeping standard used in gene expression studies.
Does RT need primer?
Primer selection. To initiate reverse transcription, reverse transcriptases require a short DNA oligonucleotide called a primer to bind to its complementary sequences on the RNA template and serve as a starting point for synthesis of a new strand.
What is a primer in RT-PCR?
A primer is a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified. Primers are also referred to as oligonucleotides.