High-quality RT-PCR with chemically modified RNA controls

Luo, Guangcheng; Zhang, Jun; Zhang, Shun; Hu, Bei; Hu, Lillian R.; Huang, Zhen

doi:10.1016/j.talanta.2020.121850

Cited by 16 publications

(10 citation statements)

References 20 publications

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“…In order to investigate the mechanism of the de novo DNA synthesis, we have explored the polymerization with the modified dNTPs (Figure ). − We have discovered that the de novo synthesis with polymerases (Pol I, Bst, and Taq functioning under 37, 63, and 72 °C, respectively) can be inhibited by the modified dNTPs (such as dNTPαSe; Figure B). Furthermore, we have found that although the polymerase generates byproduct DNAs more and faster in the presence of the nickase than its absence, dNTPαSe can inhibit the byproduct polymerization as well (Figure D).…”

Section: Resultsmentioning

confidence: 99%

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

et al. 2022

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The template-dependent DNA synthesis, with DNA polymerases, templates, and primers, is essential for disease detection, molecular biology, and biotechnology. However, DNA polymerases can also initiate de novo DNA synthesis without templates and primers, forming byproduct DNAs with random sequences. Herein, we report the mechanisms of the de novo DNA synthesis in the absence or presence of nickase by discovering the reduced bindings between the polymerases and modified dNTPs and between the nickases and the modified DNAs and finding the reduced polymerase synthesis and nickase cleavage. Furthermore, via sequencing, we have identified the mechanism of the de novo synthesis in the nickase-based isothermal amplifications, generating the random DNAs as the major byproducts. Fortunately, we have discovered a novel strategy to inhibit the undesired synthesis with the single-atom-modified nucleotides and achieved the accurate and sensitive detection of clinic samples in the isothermal amplifications. In general, we have revealed the suppression mechanisms on the de novo synthesis and demonstrated that this selenium-atom strategy can allow more accurate and sensitive detection of pathogens via the isothermal amplifications.

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Section: Resultsmentioning

confidence: 99%

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

et al. 2022

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“…Experimental methods of Quantitative real-time polymerase chain reaction (qRT-PCR) were performed as described previously (Luo et al, 2021) in accordance with the instructions provided by the manufacturer. First, total RNA was extracted from the renal cortex and GECs by TRIzol reagent.…”

Section: Quantitative Real-time Polymerase Chain Reaction Analysismentioning

confidence: 99%

Saponins from Panax notoginseng ameliorate steroid resistance in lupus nephritis through regulating lymphocyte-derived exosomes in mice

Chen

Zhou

Lu³

2022

Front. Pharmacol.

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Lupus nephritis (LN) is the most common and severe type of organ damage and an important primary disease in end-stage renal failure in patients with systemic lupus erythematosus (SLE). Clinical guidelines recommend steroid treatment, but steroid resistance has become a major factor leading to treatment failure and affecting prognosis. Our previous study demonstrated that Saponins from Panax Notoginseng (Panax ginseng saponins, PNS) could reverse steroid resistance of lymphocytes by downregulating P-glycoprotein (P-gp) expression and provide renal protection in LN mice, but the mechanism by which lymphocytes transmit these related messages to renal lamina propria cells is not clear. Therefore, we further elucidated this mechanism through holistic experiments. In this study, low-dose methylprednisolone (0.8 mg/kg/day, MP) was used to induce a steroid-resistant lupus nephritis (SR-LN) mouse model in weeks one to four, and a therapeutic steroid dosage (MP 12 mg/kg/day) or a combined PNS (PNS 100 mg/kg/day) treatment was administered from week five to eight. Lymphocyte-derived exosomes (Lyme-Exos) were isolated from the spleens of mice and injected into untreated homozygous LN mice for 14 days via the tail vein. At the end of the experiment, the efficacy and mechanism of action of different groups of Lyme-Exos on LN mice were observed. The results revealed that exogenously injected Lyme-Exos were effectively taken up by the kidney and affected the progression of kidney disease. Steroid-resistant lymphocyte-derived exosomes intervented with PNS significantly downregulated the levels of silent information regulator-related enzyme 1 (Sirt1), multidrug resistance gene 1 (MDR1), and P-gp in the renal cortex and glomerular endothelial cells (GECs); reduced serum autoantibody [antinuclear antibody (ANA) and anti-double-stranded DNA (dsDNA)] levels and inflammatory markers (WBC, PCR, and PCT); improved renal function; and attenuated urinary microalbumin excretion. Additionally, renal histopathological damage (HE staining) and fibrosis (Masson staining) were improved, and immune complex (IgG) deposition and membrane attack complex (C5b-9) production were significantly reduced; the gene levels of inflammatory factors (INF-γ, MCP-1, IL-8, IL-17, vWF, VCAM-1, IL-1β, IL-6, PTX3) in the renal cortex were downregulated. Taken together, this study showed that PNS may alleviate steroid resistance in GEC by interfering with steroid-resistant Lyme-Exos to ameliorate LN progression, which will likely provide insights into developing a new LN treatment.

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“…Most of the commercial kits adopt DNA as control in fear of RNA degradation by ribonucleases (RNases). However, DNA kits cannot report failed reverse transcriptase step, thus resulting in high false negative rates (40%) and variable sensitivity[ 3 ]. To avoid this concern, appropriate controls are necessary for viral RNA detection.…”

Section: To the Editormentioning

confidence: 99%

Comments on "Review of the role of diagnostic modalities and imaging findings in the COVID-19 pandemic"

Vulasala¹,

Gopireddy²,

Bhosale³

et al. 2022

WJR

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The present letter to the editor corresponds to the article entitled “Comprehensive literature review on the radiographic findings, imaging modalities, and the role of radiology in the coronavirus disease 2019 (COVID-19) pandemic” by Pal et al , published in World J Radiol . 2021; 13(9): 258-282. With zero to unknown prevalence, COVID-19 has created a heterogeneous and unforeseen situation across the world. Healthcare providers encountered new challenges in image interpretation, characterization, and prognostication of the disease. Pal et al delineated the radiological findings, which would guide the radiologists to identify the early signs of severe infection.

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High-quality RT-PCR with chemically modified RNA controls

Cited by 16 publications

References 20 publications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

Single-Atom-Directed Inhibition of De Novo DNA Synthesis in Isothermal Amplifications

Saponins from Panax notoginseng ameliorate steroid resistance in lupus nephritis through regulating lymphocyte-derived exosomes in mice

Comments on "Review of the role of diagnostic modalities and imaging findings in the COVID-19 pandemic"

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