Internal reference genes with the potential for normalizing quantitative PCR results for oral fluid specimens

Cheng, Ting‐Yu; Zimmerman, Jeffrey J.; Giménez-Lirola, Luis

doi:10.1017/s1466252322000044

Cited by 4 publications

(4 citation statements)

References 169 publications

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“…The relative miRNAs expression levels were measured by the △△Ct method after normalization with reference control. The U6 snRNA was the reference gene, as previously described (Isola et al, 2023) and in agreement with previous studies (Cheng et al, 2022; Donati et al, 2019; Han et al, 2014; Luo et al, 2018; Tang et al, 2019) which recommended U6 snRNA as a reference gene for miRNA quantification by RT‐PCR analysis in oral fluid specimens.…”

Section: Methodssupporting

confidence: 85%

Analysis of oral lichen planus severity on micro‐RNA linked with malignant transformation risks

Polizzi,

Santonocito,

Distefano

et al. 2023

Oral Diseases

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ObjectiveThe present study evaluated the oral tissue expression of micro‐RNA (miRNAs) linked to the potential malignant evolution of oral lichen planus (OLP). Furthermore, the correlation between OLP severity and miRNAs expression was assessed, and possible predictors of miRNAs in OLP patients were identified.MethodsThe present study enrolled 41 patients with OLP (median age 58 years) and 42 healthy controls (median age 59 years). In each patient, miRNA levels (miR‐7a‐3p,‐7a2‐3p,‐7a‐5p,‐21‐3p,‐21‐5p,‐100‐3p,‐100‐5p,‐125b‐2‐3p,‐125b‐5p,‐200b‐3p,‐200b‐5p) were assessed and analyzed through reverse transcription polymerase chain reaction. Clinical parameters and the eventual presence of OLP symptoms, signs, and disease severity scores in each patient were reported using an anamnestic questionnaire.ResultsIn comparison with healthy controls, OLP patients showed significantly higher miR‐7a‐3p,‐7a‐2‐3p,‐21‐3p, miR‐21‐5p and miR‐100‐5p levels (p < 0.05) and significantly lower miR‐125b‐2‐3p,‐125b‐5p,‐200b‐3p, and ‐200b‐5p levels (p < 0.05). Furthermore, OLP symptoms and signs and disease severity scores were significantly correlated and were also predictors of all analyzed miRNAs (p < 0.05).ConclusionsIn comparison with healthy subjects, OLP patients exhibited unbalanced oral miRNAs expression linked to the risk of potential malignant evolution of OLP. Furthermore, some miRNAs were correlated with OLP extent and were significant predictors of OLP symptoms, signs, and disease severity scores.

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Section: Methodssupporting

confidence: 85%

Analysis of oral lichen planus severity on micro‐RNA linked with malignant transformation risks

Polizzi,

Santonocito,

Distefano

et al. 2023

Oral Diseases

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“…For that reason, failure to detect the ISC would indicate a fault at some point between sample collection and final PCR testing. However, care needs to be taken in selecting ISCs because the expression of housekeeping genes varies among cell types, as a function of disease status, and by age and/or gender [ 28 , 29 , 30 , 31 ]. For example, viral infections and resultant cellular changes can affect overall gene expression [ 32 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR

Munguía-Ramírez

Armenta-Leyva

Henao-Díaz³

et al. 2023

Veterinary Sciences

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Endogenous reference genes are used in gene-expression studies to “normalize” the results and, increasingly, as internal sample controls (ISC) in diagnostic quantitative polymerase chain reaction (qPCR). Three studies were conducted to evaluate the performance of a porcine-specific ISC in a commercial porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription-qPCR. Study 1 evaluated the species specificity of the ISC by testing serum from seven non-porcine domestic species (n = 34). In Study 2, the constancy of ISC detection over time (≥42 days) was assessed in oral fluid (n = 130), serum (n = 215), and feces (n = 132) collected from individual pigs of known PRRSV status. In Study 3, serum (n = 150), oral fluid (n = 150), and fecal samples (n = 75 feces, 75 fecal swabs) from commercial herds were used to establish ISC reference limits. Study 1 showed that the ISC was porcine-specific, i.e., all samples from non-porcine species were ISC negative (n = 34). In Study 2, the ISC was detected in all oral fluid, serum, and fecal samples, but differed in concentration between specimens (p < 0.05; mixed-effects regression model). The results of Study 3 were used to establish ISC reference limits for the 5th, 2.5th and 1.25th percentiles. Overall, the ISC response was consistent to the point that failure in detection is sufficient justification for re-testing and/or re-sampling.

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“…Inter-assay repeatability was determined for each developed ELISA by testing a set of samples with known and unknown M. haemolytica antibody levels. A total of 30 samples including 5 with high antibody levels, 5 with low antibody levels, and 20 unknown samples were randomly selected from the test development trial and repeatedly tested for 4 rounds to estimate the test repeatability allowing a 10% half-width of the 95% CI of the withinsubject standard deviation (Sw) [43]. To avoid possible bias, the samples were aliquoted into 4 separate sets with randomly assigned sample ID.…”

Section: Test Repeatability Determinationmentioning

confidence: 99%

Detection of Mannheimia haemolytica-Specific IgG, IgM and IgA in Sera and Their Relationship to Respiratory Disease in Cattle

Poonsuk,

Kordik,

Hille

et al. 2023

Animals

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Mannheimia haemolytica is one of the major causes of bovine respiratory disease in cattle. The organism is the primary bacterium isolated from calves and young cattle affected with enzootic pneumonia. Novel indirect ELISAs were developed and evaluated to enable quantification of antibody responses to whole cell antigens using M. haemolytica A1 strain P1148. In this study, the ELISAs were initially developed using sera from both M. haemolytica-culture-free and clinically infected cattle, then the final prototypes were tested in the validation phase using a larger set of known-status M. haemolytica sera (n = 145) collected from feedlot cattle. The test showed good inter-assay and intra-assay repeatability. Diagnostic sensitivity and specificity were estimated at 91% and 87% for IgG at a cutoff of S/P ≥ 0.8. IgM diagnostic sensitivity and specificity were 91% and 81% at a cutoff of sample to positive (S/P) ratio ≥ 0.8. IgA diagnostic sensitivity was 89% whereas specificity was 78% at a cutoff of S/P ≥ 0.2. ELISA results of all isotypes were related to the diagnosis of respiratory disease and isolation of M. haemolytica (p-value < 0.05). These data suggest that M. haemolytica ELISAs can be adapted to the detection and quantification of antibody in serum specimens and support the use of these tests for the disease surveillance and disease prevention research in feedlot cattle.

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Internal reference genes with the potential for normalizing quantitative PCR results for oral fluid specimens

Cited by 4 publications

References 169 publications

Analysis of oral lichen planus severity on micro‐RNA linked with malignant transformation risks

Analysis of oral lichen planus severity on micro‐RNA linked with malignant transformation risks

Evaluation of a Porcine Endogenous Reference Gene (Internal Sample Control) in a Porcine Reproductive and Respiratory Syndrome Virus RT-qPCR

Detection of Mannheimia haemolytica-Specific IgG, IgM and IgA in Sera and Their Relationship to Respiratory Disease in Cattle

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