In vitro and in vivo detection of microbial gene expression in bioactivated scaffolds seeded with cyanobacteria
Abstract Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real‐time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration in vitro and in vivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein ( rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed in vitro and in vivo. Expression of16S was significantly upregulated in scaffolds in vitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to in vitro(16S: in vivo Ct value 13.21 ± 0.32, in vitro12.44 ± 0.42; rnpA in vivo Ct value 19.87 ± 0.41, in vitro17.75 ± 1.41). Overall, the results demonstrate rnpA and16S expression after 7 days of implantation in vitro and in vivo, proving the presence of living bacteria embedded in scaffolds using qPCR..
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:75 |
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| Enthalten in: |
Letters in applied microbiology - 75(2022), 2, Seite 401-409 |
| Beteiligte Personen: |
Leibrock, L.B. [VerfasserIn] |
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| BKL: |
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| Anmerkungen: |
Copyright © 2022 The Society for Applied Microbiology |
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| Umfang: |
409 |
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| doi: |
10.1111/lam.13740 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a Abstract Dermal replacement materials bioactivated with cyanobacteria have shown promising potential for wound regeneration. To date, extraction of cyanobacteria RNA from seeded scaffolds has not been described. The aim of this study was to develop a method to isolate total RNA from bioactivated scaffolds and to propose a new approach in determining living bacteria based on real‐time PCR. Transgenic Synechococcus sp. PCC 7002 (tSyn7002) were seeded in liquid cultures or scaffolds for dermal regeneration in vitro and in vivo for 7 days. RNA was extracted with a 260/280 ratio of ≥2. The small subunit of the 30S ribosome in prokaryotes (16S) and RNAse P protein ( rnpA) were validated as reference transcripts for PCR analysis. Gene expression patterns differed in vitro and in vivo. Expression of16S was significantly upregulated in scaffolds in vitro, as compared to liquid cultures, whilst rnpA expression was comparable. In vivo, both 16S and rnpA showed reduced expression compared to in vitro(16S: in vivo Ct value 13.21 ± 0.32, in vitro12.44 ± 0.42; rnpA in vivo Ct value 19.87 ± 0.41, in vitro17.75 ± 1.41). Overall, the results demonstrate rnpA and16S expression after 7 days of implantation in vitro and in vivo, proving the presence of living bacteria embedded in scaffolds using qPCR. | ||
| 700 | 1 | |a Hofmann, D.M. |4 aut | |
| 700 | 1 | |a Fuchs, B. |4 aut | |
| 700 | 1 | |a Birt, A. |4 aut | |
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| 700 | 1 | |a Frank, K. |4 aut | |
| 700 | 1 | |a Giunta, R.E. |4 aut | |
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| 700 | 1 | |a Schenck, T.L. |4 aut | |
| 700 | 1 | |a Moellhoff, N. |4 aut | |
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