 |
GeneFISH – combined detection of genes and rRNA in microorganisms
Introduction
When you are using this protocol, please cite:
Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ. Microbiol. 12: 3057-3073.
Introduction to geneFISH
A. Sample preparation
A1. Sample fixation
A2. Sample immobilization
A3. Embeding in agaroze
A4. Permeabilization
A5. Inactivation of endogeous peroxidases
B. gene detection
B1. gene probes
B1.1. probe design
B1.2 probe synthesis
B1.3 determination of hybridization conditions for hybridization and washing
B2. gene hybridization
B3. antibody binding
B4. gene CARD and inactivation of HRPs from the gene detection step
C. rRNA detection
C1. rRNA hybridization
C2. rRNA CARD
D. Mounting for microscopy and counterstaining
E. Microscopy
F. List of materials
Introduction
geneFISH is a protocol for direct linking of microbial cell identity with gene presence (Moraru et al., 2010). The geneFISH protocol comprises of three main steps:
- sample preparation (fixation, immobilization on solid support, permeabilization, inactivation of endogenous peroxidases)
- gene detection (gene hybridization with Dig labeled dsDNA polynucleotide probes, binding of anti-Dig antibody conjugated with horseradish peroxidase – HRP and catalyzed deposition of fluorescently labeled tyramides ) (see Figure 1).
- rRNA detection (rRNA hybridization with oligonucleotides probes, catalyzed reporter deposition of labeled tyramides)
The gene detection and the rRNA detection steps are interchangeable and either of them can be performed first. To minimize the DNA degradation and loss, the gene detection should be done first. However, in this case, the potentially present mRNA will be also detected. If mRNA detection interferes with the experiment, then the rRNA detection should be performed first, followed by RNA digestion and then gene detection. Since both the gene and rRNA detection steps rely on CARD reactions, it is mandatory to inactivate the HRP in between the two steps.
|
|
