Identification of a suitable qPCR reference gene in metastatic clear cell renal cell carcinoma
PBN-AR
Instytucja
Wydział Nauk Medycznych (Uniwersytet Warmińsko-Mazurski w Olsztynie)
Informacje podstawowe
Główny język publikacji
en
Czasopismo
TUMOR BIOLOGY
ISSN
1010-4283
EISSN
1423-0380
Wydawca
SPRINGER
DOI
URL
Rok publikacji
2014
Numer zeszytu
35
Strony od-do
12473-12487
Numer tomu
12
Link do pełnego tekstu
Identyfikator DOI
Liczba arkuszy
Słowa kluczowe
en
ccRCC Metastasis Reference gene RPL13 GUSB Quantitative PCR
Streszczenia
Język
en
Treść
There is no data on reference gene (RG) selection in metastatic clear-cell renal cell carcinoma (mccRCC) for quantitative PCR (qPCR) data normalization. We aimed at selecting the most stable RG for further determination of new prognostic markers. Thirty-five nonmetastatic and 35 mccRCC patients undergoing radical nephrectomy were included. Paired primary tumor (T, n = 70) and normal (C, n = 70) kidney fragments were collected; from 12 out of 35 mccRCC cases, we also collected metastasized regional lymph nodes and adrenal gland tissues (M, n = 12). After RNA extraction, reverse transcription and qPCR were performed. Samples were divided into four analyzed groups. Fifteen candidate RGs were tested by RefFinder tool and manual statistics. To present the importance of RG selection, TP53 gene expression levels in samples were normalized with the use of RG data. RPL13 gene was the most stable RG in analysis of 35 primary tumor nonmetastatic versus 35 mccRCC samples and matched metastasized T/C/M samples (n = 12, each group). GUSB was the most suitable RG in total 152 samples and in paired T and C (n = 140) kidney samples. Expression of GUSB, RPL13, and the RPL13 + RPLP0 pair were independent of clinical/sample variables. Normalization of TP53 expression levels showed variability of GAPDH and ACTB assays. GUSB or RPL13 assays should be used in mccRCC for qPCR data normalization whereas GAPDH and ACTB assays should be avoided. Prior RG studies should precede each qPCR gene expression study since RG selection is associated with the origin and proportion of specimens.
Cechy publikacji
ORIGINAL_ARTICLE
Inne
System-identifier
488658