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Statements

Subject Item
n2:RIV%2F61989592%3A15110%2F04%3A00000838%21RIV%2F2005%2FMSM%2F151105%2FN
rdf:type
skos:Concept n12:Vysledek
dcterms:description
Hsp proteins have been implicated in various cellular pathological events. We focused on expression of the Hsp70 protein family in the human hepatocyte HepG2 cell line and attempted to elaborate a method for its monitoring at the mRNA level using LightCyclerTM instrument (Roche). Cell lysates were prepared and purification of mRNA was done by either the magnetic separation method (Dynal) or using a High Pure RNA isolation kit (Roche). Corresponding cDNA was synthesized first. Six primer pairs for the Hsp70 family were examined. Among them, both primers designed in this laboratory and primers adopted from literary data were examined. Amplification reactions were effected in SYBR Green detection format. Purity and length of PCR products as well as those of the source mRNA were checked electrophoretically.As a result, an optimized primer pair giving a product of adequate degree of purity and expected chain length was selected.The method will be utilized for monitoring Hsp70 family mRNA levels in si Hsp proteins have been implicated in various cellular pathological events. We focused on expression of the Hsp70 protein family in the human hepatocyte HepG2 cell line and attempted to elaborate a method for its monitoring at the mRNA level using LightCyclerTM instrument (Roche). Cell lysates were prepared and purification of mRNA was done by either the magnetic separation method (Dynal) or using a High Pure RNA isolation kit (Roche). Corresponding cDNA was synthesized first. Six primer pairs for the Hsp70 family were examined. Among them, both primers designed in this laboratory and primers adopted from literary data were examined. Amplification reactions were effected in SYBR Green detection format. Purity and length of PCR products as well as those of the source mRNA were checked electrophoretically.As a result, an optimized primer pair giving a product of adequate degree of purity and expected chain length was selected.The method will be utilized for monitoring Hsp70 family mRNA levels in si Hsp se účastní různých patologických dějů v buňkách. Zaměřili jsme se na expresi proteinů rodiny Hsp70 v buněčné liniilidských hepatocytů HepG2. Cílem bylo vypracovat metodu pro monitorování těchto proteinů na úrovni mRNA s použitím přístroje LighCycler (Roche).Z buněčných lysátů byly purifikovány RNA magnetickou separací (souprava Dynal) nebo High Pure RNA isolation kitem(Roche). Byla syntetizována odpovídající cDNA. Pro Hsp70 jsme testovali šest párů primerů, mezi nimi dva primery navržené naší laboratoří a primery převzaté z literatury. Amplifikační reakce probíhaly v detekčním formátu SYBR Green I. Čistota a délka PCR produktů a výchozí mRNA byly ověřeny elektroforézou. Vybrali jsme optimální pár primerů, poskytující produkt odpovídajícího stupně čistoty a očekávané délky řetězce. Metoda bude využita pro monitorování hladin mRNA rodiny Hsp70 ve studiích signálních kaskád HepG2 buněk, které v současnosti v naší laboratoři probíhají.
dcterms:title
DEVELOPMENT AND OPTIMIZATION OF A REAL-TIME PCR METHOD FOR MONITORING Hsp70 PROTEIN FAMILY EXPRESSION IN HepG2 CELLS Vývoj a optimalizace real-time PCR metody pro monitorování exprese proteinů rodiny Hsp70 v HepG2 buňkách DEVELOPMENT AND OPTIMIZATION OF A REAL-TIME PCR METHOD FOR MONITORING Hsp70 PROTEIN FAMILY EXPRESSION IN HepG2 CELLS
skos:prefLabel
Vývoj a optimalizace real-time PCR metody pro monitorování exprese proteinů rodiny Hsp70 v HepG2 buňkách DEVELOPMENT AND OPTIMIZATION OF A REAL-TIME PCR METHOD FOR MONITORING Hsp70 PROTEIN FAMILY EXPRESSION IN HepG2 CELLS DEVELOPMENT AND OPTIMIZATION OF A REAL-TIME PCR METHOD FOR MONITORING Hsp70 PROTEIN FAMILY EXPRESSION IN HepG2 CELLS
skos:notation
RIV/61989592:15110/04:00000838!RIV/2005/MSM/151105/N
n3:strany
33
n3:aktivita
n7:P
n3:aktivity
P(OC B17.20)
n3:cisloPeriodika
4
n3:dodaniDat
n10:2005
n3:domaciTvurceVysledku
n9:6974899 n9:5047633 n9:5628288 n9:6087914
n3:druhVysledku
n4:J
n3:duvernostUdaju
n11:S
n3:entitaPredkladatele
n16:predkladatel
n3:idSjednocenehoVysledku
560186
n3:idVysledku
RIV/61989592:15110/04:00000838
n3:jazykVysledku
n18:eng
n3:klicovaSlova
Hsp70;real-time PCR;HepG2
n3:klicoveSlovo
n14:HepG2 n14:real-time%20PCR n14:Hsp70
n3:kodStatuVydavatele
CZ - Česká republika
n3:kontrolniKodProRIV
[29062D4EE1B0]
n3:nazevZdroje
Physiological Research
n3:obor
n13:EB
n3:pocetDomacichTvurcuVysledku
4
n3:pocetTvurcuVysledku
4
n3:projekt
n8:OC%20B17.20
n3:rokUplatneniVysledku
n10:2004
n3:svazekPeriodika
53
n3:tvurceVysledku
Červenková, Kateřina Rypka, Miroslav Uherková, Lenka Veselý, Jaroslav
s:issn
0862-8408
s:numberOfPages
1
n17:organizacniJednotka
15110