In situ hybridization: principles and practice
The workshop aims at presenting the basic steps required to design the key components and perform RNA
in situ hybridization (ISH) experiments. 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. In the following workshop, we will use 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, on the next day the signal is developed using a signal amplification strategy by the use of colorimetric substrates. The signal is then visualized by bright-field microscopy.
Additionally, the workshop will also include a demo session on the single molecule FISH (smFISH) and the TECAN EVO high throughput ISH platform. We will show the participants the advantages of smFISH as compared to colorimetric ISH in terms of quantification of RNA transcripts.
Part 2 – planned for March 2021
|March 1||||Colorimetric ISH (CISH)/Fluorescent ISH (FISH) – Dr. Joshi|
|March 2||||Introduction to single molecule FISH (smFISH) – Dr. Joshi|