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Progerin/Lamin A qPCR Detection Kit

Progerin/Lamin A qPCR Detection Kit allows two possible methods of analysis: quantitative and comparative. Two positive standard controls for lamin A and progerin are included in the kit to perform the calibration curves needed to derive the copies number of the two target transcripts in the sample. The primers included in the optimized Lamin A Master Mix recognize a specific cDNA region that is present in the human lamin A coding sequence and ab sent in human progerin coding sequence. From this amplification reaction originates a PCR produc t of 179bp. Optimized Progerin Master Mix includes primers that recognize a specific human cDNA region flanking the deletion of 150 nucleotides that characterized the progerin transcript. From this amplification reaction originates a PCR product of 160 bp. Primer s design have been accurately designed with the aim of maximizing the ability to discriminate proge rin against wild-type lamin A.


Sensibility of the assay


- LOQ, Limit Of Quantification: down to 100 lamin A and progerin copies


- LOD, Limit Of Detection: down to 10 lamin A and progerin copies


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Info

To carry out a comparative analysis of obtained data, each kit includes two further amplification mixes able to amplify short sequences belonging to two genes selected as reference genes (or housekeeping genes).

Reference genes selection was carefully performed on the basis of published data and two genes were chosen that do not have cellular functions linked to each other: beta actin and succinate dehydrogenase subunit A.

However the operator should validate the use of one or both reference genes for the samples that he intends to analyze before perform the experiment.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare and fatal genetic disease characterized by the onset of premature aging in childhood. The majority of cases is sporadic and is caused by a recurrent dominant de novo point mutation in lmna gene coding for lamin A protein. This mutation constitutively activates a cryptic site that causes the expression of a mutated form of lamin A known as progerin. The possibility to accurately quantify the mutated transcript could give to the researchers the ability to assess the effects of experimental substances in vitro administered to HGPS cells on progerin expression; moreover, researchers could also compare the amount of transcript produced in different cell lines or different growth stages and correlate the production of the mutant protein to the wild type and/or to the progression of the disease.

Recent studies have demonstrated the presence of low levels of progerin in cells and tissues from healthy subjects, and its complete absence in immortalized cells. It has been recently shown the existence of a synergistic relationship between progerin expression and progressive telomere damage during cellular senescence in healthy fibroblasts.

While it is well known that progerin production increases with cellular senescence, and thus with the number of cell passages in culture, is yet unclear whether it also correlates with the age of the donor and what is the exact role of progerin in the physiological aging.

 

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