Question

The presence and distribution of specific mRNAs within a cell can be detected by

• A. Northern Blot analysis
• B. Rnase protection assay
• C. In situ hybridization
• D. Real-time PCR

Hint:

Whenever a question asks about the "location," "distribution," or "localization" of a molecule within a cell, look for techniques that involve microscopy or the term "in situ." Techniques that start with sample homogenization (like blotting or PCR) cannot provide this information.

Answer 1

The Correct Option is C

Step 1: Problem Definition:
The task is to identify a method that detects the presence and location ("distribution") of a specific mRNA within a cell or tissue. Spatial integrity of the sample must be maintained.
Step 2: Method Analysis:
Evaluation of the techniques:
1. Northern Blot analysis: This method extracts total RNA, separates it by size, and probes for a specific mRNA. It determines mRNA presence and quantity in the sample, but spatial information is lost during cell homogenization.
2. RNase protection assay (RPA): A sensitive, solution-based method to detect and quantify specific RNAs. Like Northern blotting, it uses RNA extract and provides no spatial information.
3. In situ hybridization (ISH): "In situ" means "in its original place." This technique uses a labeled probe (DNA or RNA) complementary to the target mRNA. The probe is applied to fixed cells or tissues. It hybridizes to the target mRNA, and its location is visualized using microscopy. This reveals the spatial distribution of the mRNA within the cell or tissue, satisfying the question's requirement.
4. Real-time PCR (or qRT-PCR): Amplifies and quantifies specific RNA (after converting to cDNA) in a sample. It is quantitative, but like Northern blotting, requires RNA extraction, thus losing all spatial information.
Step 3: Solution:
In situ hybridization is the only technique that detects and localizes specific mRNAs within their cellular context.