Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

<p>Abstract</p> <p>Background</p> <p>Real-time quantitative PCR (RQ-PCR) forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC) genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (<it>ESR1</it>) was used a target gene to compare the effect of choice of EC on the estimate of gene quantity.</p> <p>Results</p> <p>The expression and validity of candidate ECs (<it>GAPDH</it>, <it>TFRC</it>, <it>ABL</it>, <it>PPIA</it>, <it>HPRT1</it>, <it>RPLP0</it>, <it>B2M</it>, <it>GUSB</it>, <it>MRPL19</it>, <it>PUM1 </it>and <it>PSMC4) </it>was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. <it>MRPL19 </it>and <it>PPIA </it>were identified as the most stable and reliable EC genes, while <it>GUSB</it>, <it>RPLP0 </it>and <it>ABL </it>were least stable. There was a highly significant difference in variance between ECs. <it>ESR1 </it>expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of <it>ESR1</it>.</p> <p>Conclusion</p> <p>We have validated two endogenous control genes, <it>MRPL19</it> and <it>PPIA</it>, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the quantitation of gene expression data, facilitating the detection of smaller changes in gene expression than otherwise possible. The combination identified here is a good candidate for use as a two-gene endogenous control in a broad spectrum of future research and diagnostic applications in breast cancer.</p

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Interactions between the estrogen receptor, its cofactors and micrornas in breast cancer

The activity of selective estrogen receptor modulators (SERMs) is not fully explained by an estrogen receptor (ER) switch model that simply turns estrogen activity on or off. A better understanding of the mechanisms involved in estrogen signaling and the development of drug resistance could help stratify patients into more coherent treatment groups and identify novel therapeutic candidates. This review describes how interactions between two novel factors known to influence estrogenic activity: nuclear receptor cofactors-protein partners which modulate estrogen action, and microRNAs-a class of recently discovered regulatory elements, may impact hormone-sensitive breast cancer. The role of nuclear receptor cofactors in estrogen signaling and the associations between ER cofactors and breast cancer are described. We outline the activity of microRNAs (miRNAs) and their associations with breast cancer and detail recent evidence of interactions between the ER and its cofactors and miRNA and provide an overview of the emerging field of miRNA-based therapeutics. We propose that previously unrecognised interactions between these two species of regulatory molecules may underlie at least some of the heterogeneity of breast cancer in terms of its clinical course and response to treatment. The exploitation of such associations will have important implications for drug development

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer-0

<p><b>Copyright information:</b></p><p>Taken from "Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer"</p><p>http://www.biomedcentral.com/1471-2199/8/107</p><p>BMC Molecular Biology 2007;8():107-107.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2211316.</p><p></p>r (Q. = E). Boxplot shows median value, interquartile-range box and outliers (*). Within gene there was no difference in gene quantities between benign and malignant tissues (> 0.05). b) Variation associated with candidate endogenous control genes and in all breast tumours relative to calibrator. Relative gene expression was calculated using the ΔCmethod and corrected for efficiency of amplification (Q. = E). There was a significant difference in variance associated with relative gene expression (= 0.001) with genes such as , , and showing greater variance than genes such as and

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer-4

<p><b>Copyright information:</b></p><p>Taken from "Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer"</p><p>http://www.biomedcentral.com/1471-2199/8/107</p><p>BMC Molecular Biology 2007;8():107-107.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2211316.</p><p></p>r (Q. = E). Boxplot shows median value, interquartile-range box and outliers (*). Within gene there was no difference in gene quantities between benign and malignant tissues (> 0.05). b) Variation associated with candidate endogenous control genes and in all breast tumours relative to calibrator. Relative gene expression was calculated using the ΔCmethod and corrected for efficiency of amplification (Q. = E). There was a significant difference in variance associated with relative gene expression (= 0.001) with genes such as , , and showing greater variance than genes such as and

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer-1

<p><b>Copyright information:</b></p><p>Taken from "Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer"</p><p>http://www.biomedcentral.com/1471-2199/8/107</p><p>BMC Molecular Biology 2007;8():107-107.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2211316.</p><p></p>nes. The gene stability value is based on the average pairwise variation between all tested genes. Low values characterise genes with greater stability, thus the x-axis from left to right indicates the ranking of the EC genes according to expression stability and the y-axis indicates the stability measure, . (b) Determination of the optimal number of ECs for normalisation. The recommended upper limit of the pairwise variation value is set at 0.15 but in meeting this criterion, sample availability, the practicality of using multiple EC genes and the degree of required resolution must be considered

Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer-3

<p><b>Copyright information:</b></p><p>Taken from "Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer"</p><p>http://www.biomedcentral.com/1471-2199/8/107</p><p>BMC Molecular Biology 2007;8():107-107.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2211316.</p><p></p>of and (Q. = E± s.e.m.). A pool of cDNA from two normal tissues was used as calibrator. There was no effect of EC on the relative quantity of in either group (> 0.05), however, there was a significant effect of EC gene on the estimate of the error associated with relative gene expression (< 0.05). The error was significantly reduced using the combination of and compared to the use of all EC genes individually with the exception of