Normalization Strategy for Selection of Reference Genes for RT-qPCR Analysis in Left Ventricles of Failing Human Hearts

Introduction: Quantitative RT-PCR is a valuable tool for assessing the gene expression in different human tissues, particularly due to its exceptional sensitivity, accuracy and reliability. However, the choice of adequate control for normalization is a crucial step, greatly affecting the results of all subsequent analyses. So far, only a few studies were focused on the selection of optimal reference genes in left ventricles of failing human hearts, leading to several disparities in experimental results focused on differential gene expression in this area. Therefore, the main objective of this study was to identify a set of suitable reference genes in normal and failing left ventricle tissues, which could increase the reliability of RT-qPCR-based studies in the future. Methods: We analyzed the expression of 15 commonly used housekeeping genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC and YWHAZ) in left ventricles of normal and failed hearts with two-step approach. In the first step, we excluded genes which are variantly expressed using ANOVA-based statistical method. Afterwards, the remaining genes were analyzed using geNorm, NormFinder and BestKeeper algorithms, together with comparative delta Cq method. Finally, the geometric mean of gene rankings across all five methods was calculated.Results: Our analysis identified IPO8, PGK1 and POLR2A as the most stably expressed genes, whereas ACB2 and B2M were found to be expressed variantly, suggesting a potential role of these genes in the pathophysiological processes in failing human hearts.Discussion/Conclusion: Using our two-step approach, we identified and validated three reference genes expressed invariantly in left ventricles of both healthy and failing human hearts, as well as provided a guideline for the selection of reference genes in studies comparing gene expression in these types of tissues.

Identification of reference genes for studies of quantitative gene expression in male and female quail tissues.

This study aimed to evaluate stability and recommend reference genes for quantitative real-time PCR in different tissues from male and female broiler quails. The stability of 10 housekeeping genes (GAPDH, RPL5, MRPS27, MRPS30, TFRC, HMBS, EEF1, LDHA, B2M and UBC) was analyzed in heart, thigh muscle, brain and spleen, by means Bestkeeper, NormFinder, GeNorm softwares with ∆Cq method. The most stable housekeeping genes were MRPS30, TFRC and HMBS in heart; MRPS30, EEF1 and HMBS in thigh muscle; B2M, GAPDH and UBC in brain; and EEF1, LDHA and HMBS in spleen. It is recommended to be used as reference genes for gene expression studies of male and female quails.

Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method

Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.

Modified CQ-Algorithms for G-Nonexpansive Mappings in Hilbert Spaces Involving Graphs

In this paper, we introduce four new iterative schemes by modifying the CQ-method with Ishikawa and [Formula: see text]-iterations. The strong convergence theorems are given by the CQ-projection method with our modified iterations for obtaining a common fixed point of two [Formula: see text]-nonexpansive mappings in a Hilbert space with a directed graph. Finally, to compare the rate of convergence and support our main theorems, we give some numerical experiments.

Characterizing Clinical Features and Creating a Gene Expression Profile Associated With Pain Burden in Children With Inflammatory Bowel Disease

Background There is often dissociation between inflammatory activity and abdominal pain in children with inflammatory bowel disease (IBD), suggesting other factors may play a role in the pain experience. Methods Patients (8 to 17 years) newly diagnosed with IBD were enrolled in the ALLAY Study: Assessing Risk Factors for Abdominal Pain in Children with Inflammatory Bowel Disease (NCT02984059). At diagnostic colonoscopy, 3 rectal biopsies were collected, and gene expression analysis was performed using Qiagen RT2 Profiler Neuropathic and Inflammatory Pain PCR Array. Relative fold difference in gene expression for 84 pain-associated genes was calculated using the 2-ΔΔ Cq method compared with pain-free controls. Factors affecting pain burden (Pain Burden Interview; PBI) were analyzed, including age, sex, rectal inflammation, and gene expression. Data were analyzed using multiple stepwise linear regression and 2-tailed t tests (P ≤ 0.05). Results Thirty-nine newly diagnosed IBD patients were included (65% male, mean age 12.75 years [SD 2.63], 23 Crohn’s disease, 16 ulcerative colitis), along with 3 controls. Mean PBI score was 7.73 (SD 6.4, range 0 to 23) for all patients. Age and sex were not predictive of pain burden, but disease activity score was (P = 0.03). Expression of TRPV3, OPRM1, P2X3, SCN9A, PTGS2, and MAPK14 were associated with PBI score. Subsequent 2-tailed t tests comparing patients with no pain (PBI score ≦ 2, N = 11) to those with pain (PBI > 2, N = 28) confirmed differential expression of TRPV3, PTGS2, and MAPK14 was in patients with pain (all P < 0.05). Conclusion Pain burden in newly diagnosed IBD patients may be linked to TRPV3, PTGS2, and MAPK14 expression, suggesting potential therapeutic targets for managing pain in IBD.

A modified Halpern-type iteration algorithm for quasi-ϕ -asymptotically nonexpansive mappings and applications

The purpose of this article is to modify the Halpern-type iteration algorithm for quasi-ϕ-asymptotically nonexpansive mapping to have the strong convergence under a limit condition only in the framework of Banach spaces. The results presented in the paper improve and extend the corresponding results of Qin et al. [Convergence of a modified Halpern-type iterative algorithm for quasi-ϕ-nonexpansive mappings, Appl. Math. Lett. 22 (2009), 1051–1055], Wang et al. [A modified Halpern-type iteration algorithm for a family of hemi-relative nonexpansive mappings and systems of equilibrium problems in Banach spaces, J. Comput. Appl. Math. 235 (2011), 2364–2371], Su et al. [Strong convergence theorems for two countable families of weak relatively nonexpansive mappings and applications, Nonlinear Anal. 73 (2010), 3890–3906], Nartinez-Yanes et al}. [Strong convergence of the CQ method for fixed point iteration processes, Nonlinear Anal. 64 (2006), 2400–2411], and others.