Question

Which type of following primers may be used in Reverse Transcriptase-PCR?

• A. oligo-dT
• B. Random
• C. Gene specific
• D. More than one answer are correct

Hint:

Use Oligo-dT if you want full-length transcripts; use random primers if your RNA is degraded; use gene-specific primers for maximum sensitivity in diagnostics.

Answer 1

The Correct Option is D

Step 1: Understanding the Concept:
Reverse Transcriptase-PCR (RT-PCR) is used to detect and quantify RNA. The first step involves converting RNA into complementary DNA (cDNA) using the enzyme reverse transcriptase. This process requires a primer to initiate DNA synthesis.

Step 2: Detailed Explanation:
There are three main strategies for priming cDNA synthesis:
1. Oligo-dT primers: These consist of a string of thymine nucleotides that hybridize to the poly-A tail found at the \(3^{\prime}\) end of most eukaryotic mRNAs. They ensure that all mRNA species are converted into cDNA.
2. Random primers (e.g., random hexamers): These are short sequences of six random nucleotides that can bind to many complementary sites across the entire length of all RNA molecules (mRNA, rRNA, tRNA). They are useful for amplifying fragmented RNA or RNAs lacking a poly-A tail.
3. Gene-specific primers: These are designed to bind specifically to a unique sequence within a target RNA. This approach is highly specific as it only converts the RNA of interest into cDNA.
All three types of primers are standard options in RT-PCR protocols depending on the objective of the study (broad profiling vs. specific target detection).

Step 3: Final Answer:
Oligo-dT, random, and gene-specific primers are all used in RT-PCR. Thus, option 4 is correct.