Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60 Co γ-rays ex vivo

Vaiphei, S. Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Chaubey, R.C.; Kma, Lakhan; Sharan, R.N.

doi:10.1093/jrr/rru074

Cited by 13 publications

(10 citation statements)

References 36 publications

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“…The employment of this method allowed us to calculate that 1 ng of the “pooled sample” contained approximatively 5822 copies of the GAPDH target (see Supporting Information Table 3). This value falls within the range found in 72 tissues (from 700 copies/ng RNA of breast tissue to 10 000 copies/ng RNA of muscle tissue) , but it is lower than those extrapolated from other papers . However, even if the absolute number (of copies) found here could be inaccurate, this is of scarce practical consequence for the aim of the present study (see below).…”

Section: Resultssupporting

confidence: 58%

Highly degraded RNA can still provide molecular information: An in vitro approach

et al. 2020

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The long‐term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR‐based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical–chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat‐mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di‐ethyl‐pyro‐carbonate‐water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde‐3‐phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r2 > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end‐point PCR of blood‐specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR‐based results show that RNA maintains the ability to be retro‐transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long‐term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.

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Section: Resultssupporting

confidence: 58%

Highly degraded RNA can still provide molecular information: An in vitro approach

et al. 2020

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“…Yet, with stability of its expression being suboptimal, it could affect the statistical outcome. Of note, preservation of GAPDH expression upon altered conditions has been reported for human umbical vein endothelial cells (HUVECs) grown under hyperglycemic conditions (Bakhashab et al 2014), chondrocytes cultured at different temperatures (Ito et al 2014) or blood cells subjected to radiation (Vaiphei et al 2015). …”

Section: Discussionmentioning

confidence: 99%

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

et al. 2016

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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.

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“…The literature shows different conclusions in relation to the housekeeping genes, for example, Vaiphei ST et al, concluded that, in blood, GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene (Vaiphei et al, 2014). On the contrary, Moreno LI et al, published that beta-2 microglobulin exhibited the highest expression for all body fluids in forensic applications (Moreno et al, 2012).…”

Section: Resultsmentioning

confidence: 99%

18S and GAPDH housekeeping genes seem to be equally useful in saliva

Jiménez-Martínez¹,

Colín-Ferreyra²,

Vilchis³

et al. 2017

REMCB

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RESUMEN.-La saliva tiene potencial para ser un instrumento de diagnóstico, asimismo se espera que su uso como herramienta de diagnóstico aumente de manera exponencial en los próximos años. Nuestro objetivo principal fue estudiar el perfil de expresión génica de dos de los genes constitutivos más comunes en saliva, gliceraldehído-3-fosfato deshidrogenasa (GAPDH) y 18S, en muestras de quince voluntarios sanos. Comparamos el valor CT, que se define como la intersección entre una curva de amplificación y una línea de umbral. De acuerdo con el análisis realizado, no hay ninguna diferencia estadísticamente significativa entre ambos genes. PALABRAS CLAVES: expresión génica, genes constitutivos, gliceraldehído-3-fosfato deshidrogenasa (GAPDH), saliva, 18S ARNr ABSTRACT.-Saliva has a potential to be a diagnostic instrument, also it is expected that its use as a diagnostic tool will increase exponentially in the next few years. Our main aim was to study the gene expression profile of two of the most common constitutive genes in saliva, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S rRNA, in samples of 15 healthy subjects. We compared the CT value, which is defined as the intersection among an amplification curve and a threshold line. According to the analysis, there is no statistically significant difference between the two genes.

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Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60 Co γ-rays ex vivo

Cited by 13 publications

References 36 publications

Highly degraded RNA can still provide molecular information: An in vitro approach

Highly degraded RNA can still provide molecular information: An in vitro approach

Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies

18S and GAPDH housekeeping genes seem to be equally useful in saliva

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