Computational protocol: Identification of Suitable Reference Genes for Gene Expression Studies of Shoulder Instability

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Protocol publication

[…] We categorized the tissue samples into the following 12 groups: 1) AI samples from cases (SI patients); 2) AS samples from cases; 3) P samples from cases; 4) all tissue samples from cases; 5) AI samples from controls; 6) AS samples from controls; 7) P samples from controls; 8) all tissue samples from controls; 9) all AI samples; 10) all AS samples; 11) all P samples; and 12) all tissue samples.For comparisons of candidate reference gene stability we used NormFinder (http://www.mdl.dk/publicationsnormfinder.htm), geNorm (http://medgen.ugent.be/~jvdesomp/genorm/http://medgen.ugent.be/~jvdesomp/genorm/), BestKeeper1 (http://www.gene-quantifcation.de/bestkeeper.html) and DataAssist (http://www.lifetechnologies.com/us/en/home/technical-resources/software-downloads/dataassist-software.html) software programs according to the recommendations of the software guides. NormFinder accounts for both intra- and inter-group variations when evaluating the stability of each single reference gene and assigns lower stability values to the genes that are most stably expressed . geNorm calculates the expression stability value (M) for each gene based on the average pairwise expression ratio between a particular gene and all other reference genes. The most stably expressed gene yields the lowest M value, and then the two most stable reference genes are determined by stepwise exclusion of the least stable gene . Bestkeeper was used to rank the 6 reference genes based on the standard deviation (SD) and coefficient of variance (CV) expressed as a percentage of the cycle threshold (Ct) level . Lastly, DataAssist software provided a metric to measure reference gene stability based on the geNorm algorithm. In contrast to the other programs, DataAssist uses RQ to calculate the stability value of individual candidate reference genes.GenEx software (http://genex.gene-quantifcation.info/) was used to determine the optimal number of reference genes by calculating the accumulated standard deviation (Acc.SD). […]

Pipeline specifications

Software tools NormFinder, geNorm, BestKeeper
Databases MedGen
Application qPCR
Organisms Homo sapiens
Diseases Wounds and Injuries