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.
What are housekeeping genes in RT-PCR?
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 examples of housekeeping genes?
Examples of housekeeping genes include actin, GAPDH and ubiquitin. Compare: luxury gene. See also: genetic expression, transcription.
How many housekeeping genes are needed for qPCR?
It is recommended to always use at least two reference genes, since the use of only one may lead to relatively large errors (Nicot et al. 2005).
What is the best housekeeping gene?
The top 3 most stable housekeeping genes were: UBC, SDHA and ARBP for all states. Following statistical analysis using the Mann-Whitney-U Test, only UBC and SDHA for the 24 hr ligated, 2 wk ligated and 2 wk ligated + 3 day de-ligated states showed no change from the unoperated control group (>0.05) (Figure 4).
What is a good housekeeping gene?
What is a Good Housekeeping Gene? The definition of a good housekeeping gene, is one that has stable expression in varying conditions, thus can be used to normalize other influences which may skew the result.
What do housekeeping genes do?
Housekeeping genes are typically constitutive genes that are required for the maintenance of basal cellular functions that are essential for the existence of a cell, regardless of its specific role in the tissue or organism.
What is the difference between housekeeping gene and reference gene?
housekeeping genes are typically constitutive genes that are required for the maintenance of basic cellular function, and are expressed in all cells of an organism under normal and patho-physiological conditions. Reference genes are expressed in all cells of an organism under normal and patho-physiological conditions.
How many housekeeping genes are there?
Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. ], many more than the dozen or so commonly used control genes. Eisenberg E. Levanon E.Y.
Why do we use housekeeping genes as internal controls in RT-PCR?
On the other hand Housekeeping genes are widely used as internal controls for experimental studies 1. The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification.
What is real time PCR?
Real time PCR is one of the most sensitive and reproducible quantification methods for gene expression analysis. It provides simultaneous measurement of gene expression in many different samples for a number of genes.
How can I improve the reliability of my relative RT-PCR experiment?
The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in RT-PCR efficiency and errors in sample quantification.
What are the best housekeeping genes found with geNORM?
hsp20.2 gene expression during late blight exposure normalized with 18S rRNA as the internal control showed similar results to those obtained with ef1α or with the two housekeeping genes used. In this experiment, 18S rRNA belonged to the two best housekeeping genes found with geNorm software as opposed to the other stress experiments.