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. 2000 May;66(5):1796-800.
doi: 10.1128/aem.66.5.1796-1800.2000.

In Situ Reverse transcription-PCR for Monitoring Gene Expression in Individual Methanosarcina Mazei S-6 Cells

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In Situ Reverse transcription-PCR for Monitoring Gene Expression in Individual Methanosarcina Mazei S-6 Cells

M Lange et al. Appl Environ Microbiol. .
Free PMC article

Abstract

An in situ reverse transcription-PCR protocol for detecting specific mRNA in Methanosarcina mazei S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene dnaK. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate-Fast Red detection system and binding of anti-digoxigenin-alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the dnaK transcript.

Figures

FIG. 1
FIG. 1
Analysis of supernatant fractions by using DMSO, heat-cold cycling, or no treatment for permeabilization. Ten-microliter portions of the supernatants from PCR mixtures were electrophoresed in an agarose gel and stained with ethidium bromide. (A) Supernatants from the first round of PCR. (B) Supernatants from the second round of PCR. The treatments used are indicated below the lane numbers. Abbreviations: H.S., heat shock at 45°C for 30 min; PCR1, first round of PCR (performed with primers dnaKf and dnaKr), including an RT step and 20 cycles of standard amplification; PCR2, second round of PCR (performed with primers dnaKf and dnaKi) with 5 or 20 cycles as indicated (for the samples in lanes 15 through 17 RT was included); Perm., type of permeabilization treatment (D, DMSO treatment; T, heat-cold treatment; N, no permeabilization treatment); +, used; −, not used.
FIG. 2
FIG. 2
Detection of dnaK in M. mazei S-6 cells. Cells were permeabilized by thermal cycling and subjected to RT-PCR and a seminested PCR by using 10 cycles in the second round. See text for details. (A and C) Phase-contrast photomicrographs. (B and D) Epifluorescence photomicrographs. The cells in panels A and B were heat shocked for 30 min at 45°C.
FIG. 3
FIG. 3
Cell permeabilization with lysozyme. Fixed cells were treated with lysozyme at concentrations of 0.01 mg/ml (lanes 1 through 4), 0.1 mg/ml (lanes 5 through 8), 0.5 mg/ml (lanes 9 through 12), 1.0 mg/ml (lanes 13 through 16), or 3.0 mg/ml (lanes 17 through 20). Ten-microliter portions of supernatants obtained from the first round of PCR (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, and 18) or the second round of PCR (lanes 3, 4, 7, 8, 11, 12, 15, 16, 19, and 20) were electrophoresed in an agarose gel. Odd lanes contained samples from cells that had not been heat shocked. Even lanes contained samples from cells that had been heat shocked at 45°C for 30 min.
FIG. 4
FIG. 4
Cells of M. mazei were fixed and treated with DNase as described in the text. Ten microliters of the PCR product was visualized in a stained agarose gel; all lanes are from same gel, and irrelevant lanes were removed. Lanes 1 and 2 are replicates, and lanes 3 and 4 are replicates. After DNase treatment the cells in lanes 1 and 2 were subjected to RT followed by PCR; the RT step was omitted for the cells in lanes 3 and 4. Lane 5 contained a no-polymerase control, which allowed us to judge background staining.

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