Online workshop
In situ hybridization: principles and practice part II
FISH
July 2021
The second part of the workshop aims at showing the basic steps at doing Fluorescent in situ hybridization (FISH) The tutors come from a leading EU lab at the Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, which specializes in high-throughput ISH.
RNA ISH is a qualitative technique that requires a labelled complementary RNA probe to localize a specific RNA in tissues or cells. The RNA probe can be labelled with either a radio-, fluorescent, or an antigen base. As shown in the first workshop, we have used an antigen (e.g., Digoxigenin) labelled RNA probe to detect specific genes in the monkey brain tissue. In the first step, gene specific primers are designed to amplify the gene of interest to be studied. These are flanked with T7- and Sp6- RNA polymerase binding sites on the forward and reverse primers respectively. The DNA is amplified from the cDNA pool and the resulting product is called the 'template DNA', which will be subsequently used for RNA probe synthesis. The template DNA is sequenced using T7 and Sp6 primers to verify correct amplification of target gene.
Following sequencing, we carry out in vitro transcription (IVT) of the template DNA using Digoxigenin labelled UTPs and Sp6 RNA polymerase. The resulting product will be antisense RNA probe that is complementary to our target RNA of interest. Next hybridization is carried out on 20-µm thick fresh frozen tissue sections at 60oC overnight. After hybridization, the signal is developed using a signal amplification strategy using Tyramide Signal Amplification which provides up to 100-fold sensitivity. The signal is then visualized by fluorescent microscopy. Instead of Alkaline phosphatase used in the colorimetric detection, as shown in the first seminar, here we are using peroxidase (POD) to catalyse covalent deposition of fluorophores. In addition, one may continue the protocol with immunofluorescence.
Program
Part 2 –July 7-8, 2021 | | |
July 7 |
11:00h |
Prof. Dr. Anton Tonchev - Opening. Dr. Martin Ivanov – Workshop overview and schedule of experiments Dr. Parth Joshi
- Introduction to the method
Dr. Parth Joshi and Dr. Martin Ivanov – First day of the protocol |
July 8 |
11:00h | Dr. Parth Joshi and Dr. Martin Ivanov – Second day of the protocol Dr. Parth Joshi and Dr. Martin Ivanov- Q&A session End of the workshop |