Tips to Follow for Conducting a Successful ISH

RNA

ISH also stands for in situ hybridization. This is a technique for detecting the specific RNA and DNA sequence in the tissue. The process gets done by labeling DNA or RNA probe. The probe, in general, gets detected by using an antibody. The method plays a crucial role in many different clinical settings and scientific research. It can also be used as a diagnostic tool to understand the gene mapping, gene expression, cytogenetics and prenatal diagnosis & development.

The detection process of a particular nucleotide sequence gets completed by using a complementary strand. This type of complementary strand gets hybridized with a probe. The hybrids are visualized by using an autoradiography for probes (radioactively tagged). The process of ISH offers plenty of advantages to the researchers. For example, it enables them to study the distribution of nucleotides. It helps them to understand the particular gene sequence of a target gene or protein.

Two of the primary methods of the ISH in order to visualize the DNA and RNA are:

  1. FISH (Fluorescent in situ hybridization)
  2. CISH (Chromogenic in situ hybridization)

In both the processes, researchers use the particular nucleic acid probes. These probes are hybridized with the sample of the tissue. Researchers can get to see it either with a fluorescent microscopy or by using a bright field microscopy. In both the methods of CISH and FISH, the preparation of the tissue and process of adding nucleotide probes are same. However, the probe detection process may vary than each other. Researchers can use a wide range of probes in this technique from locus specific probes to telomere repeats from centromere repeats to whole chromosomes.

Protocol for In Situ Hybridization (ISH)

In the below section you will come to know about the standard protocol of ISH that will include tissue preparation, hybridization and the addition of probe.

Preparation of Tissues

  1. Ensure that the tissues you are using for the research are paraffin embedded or frozen. However, it is always better to use frozen tissues that were preserved in nucleic acids.
  2. You can also consider using formalin fixed tissues
  3. Cut the tissues at 0.4 or 0.4 µm thick before inserting it on the slide.
  4. Wash sample thoroughly with ethanol or xylene

Adding Probe

  1. Use only synthetic oligonucleotide, cRNA or cDNA as a probe. An RNA probe needs to hybridize with compatible mRNA. On the other hand, the DNA probe needs to be hybridized with the corresponding cDNA.
  2. Ensure that the probe is 40 to 1000 bases long
  3. Denaturation occurs at 95° Celsius – keep that in mind
  4. Select the probe based on other specific need and the sensitivity.
  5. You can label the probe with both non-isotope and isotope

Hybridization

  1. Consider adding dextran sulfate to increase the hydration of the specific sample. It will help you to increase the hybridization rate.
  2. You can also use DTT (dithiothreitol) or formamide to conduct hybridization even at a lower temperature.
  3. You can add sodium chloride and sodium citrate (which is also known as SSC) to reduce the rate of electrostatic attraction between strands.

By following the above tips you will be able to conduct a successful in situ hybridization.

 

Author’s Bio: Tom Clark is a renowned science based research writer who has a special interest in hybridization. However, he has also published plenty of journals on total RNA isolated from hard to obtain tissues like a cancer tumor, hypertension, liver cirrhosis, lupus, and Alzheimer’s patients.

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