Uncovering the stability status of the reputed reference genes in breast and hepatic cancer cell lines

Accurate and reliable relative gene expression analysis via the Reverse Transcription-quantitative Real Time PCR (RT-qPCR) method strongly depends on employing several stable reference genes as normalizers. Utilization of the reference genes without analyzing their expression stability under each experimental condition causes RT-qPCR analysis error as well as false output. Similar to cancerous tissues, cancer cell lines also exhibit various gene expression profiles. It is crucial to recognize stable reference genes for well-known cancer cell lines to minimize RT-qPCR analysis error. In this study, we showed the expression level and investigated the expression stability of eight common reference genes that are ACTB, YWHAZ, HPRT1, RNA18S, TBP, GAPDH, UBC, and B2M, in two sets of cancerous cell lines. One set contains MCF7, SKBR3, and MDA-MB231 as breast cancer cell lines. Another set includes three hepatic cancer cell lines, including Huh7, HepG2, and PLC-PRF5. Three excel-based softwares comprising geNorm, BestKeeper, and NormFinder, and an online tool, namely RefFinder were used for stability analysis. Although all four algorithms did not show the same stability ranking of nominee genes, the overall results showed B2M and ACTB as the least stable reference genes for the studied breast cancer cell lines. While TBP had the lowest expression stability in the three hepatic cancer cell lines. Moreover, YWHAZ, UBC, and GAPDH showed the highest stability in breast cancer cell lines. Besides that, a panel of five nominees, including ACTB, HPRT1, UBC, YWHAZ, and B2M showed higher stability than others in hepatic cancer cell lines. We believe that our results would help researchers to find and to select the best combination of the reference genes for their own experiments involving the studied breast and hepatic cancer cell lines. To further analyze the reference genes stability for each experimental condition, we suggest researchers to consider the provided stability ranking emphasizing the unstable reference genes.

Validation of Reference Genes for Quantitative PCR in Johnsongrass (Sorghum halepense L.) under Glyphosate Stress

Weeds are one of the main causes of the decrease in crop yields, with Johnsongrass (Sorghum halepense L.) being one of the most significant. Weeds can be controlled by herbicides, but some have developed resistance. Quantitative PCR is the technique of choice for studying gene expression related to herbicide resistance because of its high sensitivity and specificity, although its quantitative accuracy is highly dependent on the stability of the reference genes. Thus, in this study we evaluated the stability of different reference genes of glyphosate-resistant S. halepense. Nine genes frequently used as reference genes were selected: MDH, ADP, PP2A, EIF4α, ACT, ARI8, DnaJ, Hsp70, and ALS1, and their expression analyzed in susceptible and resistant biotypes at 0, 24 and 72 h post-application of glyphosate. The stability was analyzed with the geNorm, NormFinder, and BestKeeper software programs and using the ΔCt method. RefFinder was used to generate a comprehensive stability ranking. The results showed that PP2A and ARI8 were the most stable genes under the test conditions. EPSPS expression was also verified against the best two and the worst two reference genes. This study provides useful information for gene expression analysis under glyphosate stress and will facilitate resistance mechanism studies in this weed species.

Hyaluronan Hydrogels for Injection in Superficial Dermal Layers: An In Vitro Characterization to Compare Performance and Unravel the Scientific Basis of Their Indication

Background: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. Methods: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. Results: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels’ ability to prompt elastin expression and in ColIV/ColI ratio. Conclusions. A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels’ behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.

Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae)

Aquatica leii Fu and Ballantyne is a species of rare aquatic firefly and endemic in China. It is considered good material to study the molecular mechanism of sexual flash communication systems. To improve conservation and behavioral research strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been very few reports on appropriate reference genes in any species of firefly. Here, we evaluated eight widely utilized reference genes including 18S, Actin, Reep5, Odc1, Tub, Gapdh, Ef1a and S27Ae for their expression stabilities in A. leii under three different conditions, i.e., life stage, tissue and dsRNA injection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend S27Ae and Reep5 as the most appropriate reference genes for molecular studies in different life stages; Ef1a and Odc1 for different tissues; Tub and Odc1 for RNAi studies. The most appropriate reference genes in all treatments are S27Ae and Tub. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis.

Identification and validation of appropriate reference genes for gene expression analysis in Schima superba

Background: Real-time quantitative PCR (RT-qPCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results: Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions: This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

H2S Stability of Metal-Organic Frameworks: A Computational Assessment

<p>The H₂S stability of a range of MOFs was systematically assessed by first-principle calculations. The most likely degradation mechanism was first determined and we identified the rate constant of the degradation reaction as a reliable descriptor for characterizing the H₂S stability of MOFs. A qualitative H₂S stability ranking was thus established for the list of investigated materials. Elemental structure-stability relationships were further envisaged considering several variables including the nature of the linkers and their grafted functional groups, the pore size, the nature of metal sites and the presence/nature of coordinatively unsaturated sites. This knowledge enabled the anticipation of the H₂S stability of one prototypical MOF, e.g. MIL-91(Ti), which has been previously proposed as a good candidate for CO₂ capture. This computational strategy enables an accurate and easy handling assessment of the H₂S stability of MOFs and offers a solid alternative to experimental characterizations that require the manipulation of a highly toxic and corrosive molecule. </p>

H2S Stability of Metal-Organic Frameworks: A Computational Assessment

<p>The H₂S stability of a range of MOFs was systematically assessed by first-principle calculations. The most likely degradation mechanism was first determined and we identified the rate constant of the degradation reaction as a reliable descriptor for characterizing the H₂S stability of MOFs. A qualitative H₂S stability ranking was thus established for the list of investigated materials. Elemental structure-stability relationships were further envisaged considering several variables including the nature of the linkers and their grafted functional groups, the pore size, the nature of metal sites and the presence/nature of coordinatively unsaturated sites. This knowledge enabled the anticipation of the H₂S stability of one prototypical MOF, e.g. MIL-91(Ti), which has been previously proposed as a good candidate for CO₂ capture. This computational strategy enables an accurate and easy handling assessment of the H₂S stability of MOFs and offers a solid alternative to experimental characterizations that require the manipulation of a highly toxic and corrosive molecule. </p>

Performance Evaluation and Shelf Life of Onion Varieties in Western Tigray, Ethiopia

Onion (Allium cepa L.) is an important underground vegetable bulb crop of tropical and subtropical part of the world. The experiment was carried out in randomized complete block design (RCBD) with three replications and conducted during 2016 to 2018 under irrigation condition at three testing sites. The experiment was designed to select high yielding and good shelf life varieties with the following specific objectives (1) to evaluate and select high yielding onion varieties, (2) to select the adaptable onion varieties and (3) to evaluate and select onion varieties having long shelf life. The study result showed that different variations were observed among the varieties; (29.82%) by environments, (21.94%) Genotype x Environment Interaction and genotypes (6.6. The high environmental variations and differential response of genotypes to the variable environments leading to inconsistency ranking of genotypes across years and locations. The highest yield was obtained from Bombey red (440.3 kgha-1), followed by Nafis(395 kgha-1), Shendi(391 kgha-1), Nasik red(373 kgha-1) and Adama red(387 kgha-1) respectively. The highest %yield loss recorded from Adama red (28.30%) followed by Nafis (23.23%), Nasik red (22.90) Bombey red (22.48%) and Shendi (5.67%) respectively. Even though, the stability ranking of the varieties were varied among the stability parameters. Nafis and Bombey red were found the most stable. While Nasik red, Adama red and Shendi unstable across years and locations. From each of the onion varieties 5 kg was stored for three months and shelf life data recorded and analyzed using R software. Based on the study Shendi can store more than six months without any problem will be recommended and promoted for commercial production. Therefore, the stable varieties recommended and promoted for wider areas.

Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

Quantitative real time polymerase chain reaction (qPCR) data are normalised using endogenous control genes. We aimed to: (1) demonstrate a pathway to identify endogenous control genes for qPCR analysis of formalin-fixed paraffin-embedded (FFPE) tissue using bladder cancer as an exemplar; and (2) examine the influence of probe length and sample age on PCR amplification and co-expression of candidate genes on apparent expression stability. RNA was extracted from prospective and retrospective samples and subject to qPCR using TaqMan human endogenous control arrays or single tube assays. Gene stability ranking was assessed using coefficient of variation (CoV), GeNorm and NormFinder. Co-expressed genes were identified from The Cancer Genome Atlas (TCGA) using the on-line gene regression analysis tool GRACE. Cycle threshold (Ct) values were lower for prospective (19.49 ± 2.53) vs retrospective (23.8 ± 3.32) tissues (p < 0.001) and shorter vs longer probes. Co-expressed genes ranked as the most stable genes in the TCGA cohort by GeNorm when analysed together but ranked lower when analysed individually omitting co-expressed genes indicating bias. Stability values were < 1.5 for the 20 candidate genes in the prospective cohort. As they consistently ranked in the top ten by CoV, GeNorm and Normfinder, UBC, RPLP0, HMBS, GUSB, and TBP are the most suitable endogenous control genes for bladder cancer qPCR.

Atypical structures of GAA/TTC trinucleotide repeats underlying Friedreich’s ataxia: DNA triplexes and RNA/DNA hybrids

Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich’s ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.