A spiking strategy facilitates housekeeping selection for RT-qPCR analysis under different biotic stresses in eggplant.
Endogenous housekeeping genes are traditionally employed to normalize the expression of target genes in RT-qPCR studies. Assuming that a perfect housekeeping suitable for every condition does not exist, expression stability of the chosen reference gene should be evaluated at every new experiment. Th...
| Publicado en: | Protoplasma Vol. 254; no. 6; pp. 2215 - 2224 |
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| Autores principales: | , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Springer Nature
Nov2017
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=125839849&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 125839849 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 0033183X 2CF jtl: Protoplasma issn: 0033183X maglogo: N pubinfo: dt: Nov2017 vid: 254 iid: 6 pid: 237 pub: Springer Nature place: New York, New York artinfo: ui: 125839849 143904197 10.1007/s00709-017-1111-2 125839849 ppf: 2215 ppct: 9 formats: fmt: @attributes: type: P tig: atl: A spiking strategy facilitates housekeeping selection for RT-qPCR analysis under different biotic stresses in eggplant. aug: au: Barbierato, Valeria Sala, Tea Rinaldi, Paola Bassolino, Laura Barchi, Lorenzo Rotino, Giuseppe Toppino, Laura affil: CREA- Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Unità di Ricerca per l'Orticoltura , Via Paullese, 28 Montanaso Lombardo 26836 Lodi Italy sug: ab: Endogenous housekeeping genes are traditionally employed to normalize the expression of target genes in RT-qPCR studies. Assuming that a perfect housekeeping suitable for every condition does not exist, expression stability of the chosen reference gene should be evaluated at every new experiment. The housekeeping selection process reveals furthermore complicated and time-consuming when different conditions have to be compared in the same experimental dataset. As an alternative strategy, we spiked an external reference transcript (ERT) into all RNA samples of our dataset (eggplant roots subjected to different biotic stresses), and used it to normalize the expression levels of native candidate housekeeping. ERT expression resulted highly stable across all samples and enabled to indicate glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) as the most stable endogenous housekeeping. This result was confirmed by the use of GeNorm, Normfinder, and BestKeeper algorithms. This method might be generally applied to expedite the selection process of the best reference gene. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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