Abstract
Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A, TP53, and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA, RPLP0, YWHAZ, and IPO8 were repeatedly highly ranked while ACTB, B2M, UBC, and PGK1 were ranked poorly. Normalization against PPIA/RPLP0/SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.
Highlights
Real-time reverse transcription PCR (RT-qPCR) is frequently employed for unravelling the pathomechanisms of diseases to aid the research on new potential biomarkers and therapeutic strategies (Bustin and Murphy 2013)
Standardization against inappropriate housekeeping genes (HKG) may lead to invalid conclusions when much more sensitive assays like quantitative real-time PCR are used as shown by Caradec et al (2010) demonstrating a false PAR1 up-regulation in LNPCaP cells grown in response to hypoxia following normalization against unstable HKG
We evaluated the following HKG: ACTB, B2M, glyceraldehade-3-phosphate dehydrogenase (GAPDH), GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ
Summary
Real-time (quantitative) reverse transcription PCR (RT-qPCR) is frequently employed for unravelling the pathomechanisms of diseases to aid the research on new potential biomarkers and therapeutic strategies (Bustin and Murphy 2013). Glyceraldehade-3-phosphate dehydrogenase (GAPDH), the most frequently used normalizer, has been demonstrated to increase over 40-fold in severe sepsis (Cummings et al 2014) but decrease with ageing (Vigelsø et al 2015). Alterations in HKG expression may be too subtle to affect the results obtained by semi-quantitive methods like end-point PCR or to manifest themselves at protein level. Standardization against inappropriate HKG may lead to invalid conclusions when much more sensitive assays like quantitative real-time PCR are used as shown by Caradec et al (2010) demonstrating a false PAR1 up-regulation in LNPCaP cells grown in response to hypoxia following normalization against unstable HKG. A necessity of HKG validation for various experimental settings, if RT-qPCR is to be used, is increasingly recognized
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