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The key criterion for the use of a housekeeping gene in this manner is that the chosen housekeeping gene is uniformly expressed with low variance under both control and experimental conditions. Validation of housekeeping genes should be performed before their use in gene expression experiments such as RT-PCR.
The guideline consists of the following elements: 1) experimental design, 2) sample, 3) nucleic acid extraction, 4) reverse transcription, 5) qPCR target information, 6) oligonucleotides, 7) protocol, 8) validation, and 9) data analysis. Specific items within each element carry a label of either E (essential) or D (desirable).
SYBR Green fluorescence chart produced in real-time PCR Melting curve produced at the end of real-time PCR. A real-time polymerase chain reaction (real-time PCR, or qPCR when used quantitatively) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).
HRT Atlas allows searching of a complete list of reliable candidate reference genes and transcripts for RT-qPCR normalization in more than 120 human and mouse tissues or cell types. The database also offers some empirically validated primers and predicted modifiers (disease and small molecules) of the expression of these reference genes.
The MIQE guidelines were created due to the low quality of qPCR data submitted to academic journals at the time, which was only becoming more common as Next Generation Sequencing machinery allowed for such experiments to be run for a cheaper cost. Because the technique is utilized across all of science in multiple fields, the instruments ...
Due to the sequence conservation in housekeeping genes, MLST sometimes lacks the discriminatory power to differentiate bacterial strains, which limits its use in epidemiological investigations. To improve the discriminatory power of MLST, a multi-virulence-locus sequence typing (MVLST) approach has been developed using Listeria monocytogenes ...
The inclusion of housekeeping genes, whose expression should be constant under the conditions, is used for normalisation. The most commonly used house keeping genes include GAPDH and α-actin, although the reliability of normalisation through this process is questionable as there is evidence that the level of expression can vary significantly. [11]
[71] [72] qPCR is limited in its ability to precisely quantify gene amplifications in several diseases, including Crohn’s disease, HIV-1 infection, and obesity. [73] [69] [72] dPCR was designed to measure the concentration of a nucleic acid target in copies per unit volume of the sample.