Rpp30 Reference Gene
Abigail Tyrie
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Ribonuclease P protein subunit p30 (RPP30) is a highly conserved housekeeping gene that exists in many species and tissues throughout the three life kingdoms (archaea, bacteria, and eukaryotes). RPP30 is closely related to a few types of tumors in human diseases but has a very stable transcription level in most cases. Based on this feature, increasing number of studies have used RPP30 as an internal reference gene. Here, the structure and basic functions of RPP30 are summarized and the likely relationship between RPP30 and various diseases in plants and human is outlined. Finally, the current application of RPP30 as an internal reference gene and its advantages over traditional internal reference genes are reviewed. RPP30 characteristics suggest that it has a good prospect of being selected as an internal reference; more work is needed to develop this research avenue.
Background Anemia and retinopathy of prematurity (ROP) are common comorbidities experienced by preterm infants, yet the role of anemia on the pathogenesis of ROP remains unclear. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for estimating the gene expression changes at the transcript level but requires identification of stably expressed reference genes for accurate data interpretation. This is particularly important for oxygen induced retinopathy studies given that some commonly used reference genes are sensitive to oxygen. This study aimed to identify stably expressed reference genes among eight commonly used reference genes in the neonatal rat pups' retina upon exposure to cyclic hyperoxia-hypoxia, anemia, and erythropoietin administration at two age groups (P14.5 and P20) using Bestkeeper, geNorm, and Normfinder, three publicly available, free algorithms, and comparing their results to the in-silico prediction program, RefFinder. Results The most stable reference gene across both developmental stages was Rpp30, as predicted by Genorm, Bestkeeper, and Normfinder. RefFinder predicted Tbp to be the most stable across both developmental stages. At P14.5, stability varied by prediction program; at P20, RPP30 and MAPK1 were the most stable reference genes. Gapdh, 18S, Rplp0, and HPRT were predicted as the least stable reference genes by at least one of the prediction algorithms. Conclusion Expression of Rpp30 is the least affected by experimental conditions of oxygen induced retinopathy, phlebotomy induced anemia and erythropoietin administration at both timepoints of P14.5 and P20.
N2 - Background Anemia and retinopathy of prematurity (ROP) are common comorbidities experienced by preterm infants, yet the role of anemia on the pathogenesis of ROP remains unclear. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for estimating the gene expression changes at the transcript level but requires identification of stably expressed reference genes for accurate data interpretation. This is particularly important for oxygen induced retinopathy studies given that some commonly used reference genes are sensitive to oxygen. This study aimed to identify stably expressed reference genes among eight commonly used reference genes in the neonatal rat pups' retina upon exposure to cyclic hyperoxia-hypoxia, anemia, and erythropoietin administration at two age groups (P14.5 and P20) using Bestkeeper, geNorm, and Normfinder, three publicly available, free algorithms, and comparing their results to the in-silico prediction program, RefFinder. Results The most stable reference gene across both developmental stages was Rpp30, as predicted by Genorm, Bestkeeper, and Normfinder. RefFinder predicted Tbp to be the most stable across both developmental stages. At P14.5, stability varied by prediction program; at P20, RPP30 and MAPK1 were the most stable reference genes. Gapdh, 18S, Rplp0, and HPRT were predicted as the least stable reference genes by at least one of the prediction algorithms. Conclusion Expression of Rpp30 is the least affected by experimental conditions of oxygen induced retinopathy, phlebotomy induced anemia and erythropoietin administration at both timepoints of P14.5 and P20.
AB - Background Anemia and retinopathy of prematurity (ROP) are common comorbidities experienced by preterm infants, yet the role of anemia on the pathogenesis of ROP remains unclear. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) is a sensitive technique for estimating the gene expression changes at the transcript level but requires identification of stably expressed reference genes for accurate data interpretation. This is particularly important for oxygen induced retinopathy studies given that some commonly used reference genes are sensitive to oxygen. This study aimed to identify stably expressed reference genes among eight commonly used reference genes in the neonatal rat pups' retina upon exposure to cyclic hyperoxia-hypoxia, anemia, and erythropoietin administration at two age groups (P14.5 and P20) using Bestkeeper, geNorm, and Normfinder, three publicly available, free algorithms, and comparing their results to the in-silico prediction program, RefFinder. Results The most stable reference gene across both developmental stages was Rpp30, as predicted by Genorm, Bestkeeper, and Normfinder. RefFinder predicted Tbp to be the most stable across both developmental stages. At P14.5, stability varied by prediction program; at P20, RPP30 and MAPK1 were the most stable reference genes. Gapdh, 18S, Rplp0, and HPRT were predicted as the least stable reference genes by at least one of the prediction algorithms. Conclusion Expression of Rpp30 is the least affected by experimental conditions of oxygen induced retinopathy, phlebotomy induced anemia and erythropoietin administration at both timepoints of P14.5 and P20.
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The molecular diagnosis of COVID-19 by RT-PCR detection of SARS-CoV-2 RNAs has been flawed by the problem of false negative results. As any other PCR-based diagnostic method, many factors such as the quality of test kits and specimens may reduce the sensitivity and specificity of the test. An in-depth investigation is needed to identify the major causes for false negative results. In this study, we tried to gain insights by retrospectively analyzing test results and data from carefully matched patient specimens and sequential specimens from patients with conflicting test results.
Our analyses demonstrated that clinical specimens with high cellular contents, measured by the RNA levels of a housekeeping gene ribonuclease P/MRP subunit p30 (RPP30), provided a significantly higher positive rate in SARS-CoV-2 detection. In fact, the detectability of SARS-CoV-2 RNAs in respiratory tract specimens was found to be highly associated with cellular content, represented by RPP30 RNA levels. Based on data from these specimens, a set of Ct cutoff values for RPP30 RT-PCR reaction was established for specific tests based on amplification of specific target regions using specific types of specimens. These Ct cutoff values from RPP30 RT-PCR could be used as criteria for identification of false negative results. Our data also suggest that false negative results accounted for most contradicting test results in SARS-CoV-2 RNA detection in clinical use due to poor specimen quality. Thus, the quality of specimens, defined by the cellular content, is a key factor for satisfactory RT-PCR results and could be assessed by an internal reference such as RPP30.
A total of 161 confirmed COVID-19 patients admitted into the Second Hospital of Fuyang from January 20 to February 29, 2020 were enrolled in this retrospective single-center study. The diagnosis of COVID-19 for patients was performed according to the Guidelines for the Diagnosis and Treatment of New Coronavirus Pneumonia (version 7) published by the National Health Commission of China. The study was approved by the Ethics Committee of the Second Hospital of Fuyang (Number 2020020004). Written informed consents were obtained from all patients.
Sputum and/or throat-swab specimens were collected from patients and maintained in viral-transport medium prior to laboratory testing. The RNA was extracted with a Nucleic Acid Extraction Kit (Shuoshi Ltd, Taizhou, China), The RT-PCR reactions for all three gene fragments were conducted in the same reaction tube on a SLAN 96P Fluorescence Quantitative PCR Amplification Instrument (Hongshi Ltd, Shanghai, China) by following the manual of the kit (Bojie Ltd, Shanghai, China). As recommended by the Guidelines for Diagnosis and Treatment of the New Coronavirus Pneumonia published by the National Health Commission of China, a test result was considered positive if the Ct values of both target region amplifications of SARS-CoV-2 genome in one test were lower than 38, or the Ct value in one of two target region amplifications was lower than 38 in two consecutive tests.
SARS-CoV-2 RT-PCR may fail to provide a positive result with specimens from COVID-19 patients. Sputum and throat swabs specimens are commonly used for RNA extraction and RT-PCR detection of SARS-CoV-2 RNA. A retrospective analysis showed that only 39.1% of 573 sputum specimens and 19.2% of 579 throat specimens from COVID-19 patients collected at different time points were consistently tested positive for SARS-CoV-2 RNAs by RT-PCR (Fig. 1A, 1B). Many samples were positive for only the reaction detecting the N region of SARS-CoV-2 RNA. Taking advantage of access to data for a large number of specimens from a cohort of 161 confirmed patients, we systematically analyzed the data to gain insights into the causes for questionable results and find the right solutions.
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