Difference Between Random Primers and Oligo dT

The key difference between random primers and oligo dT is that the random primer is a mixture of all possible hexamer oligonucleotide sequences, while the oligo dT primer consists of a single-stranded stretch of 12–18 deoxythymidines.

Reverse transcription is a mechanism in which cDNA can be synthesized using mRNA or any species of RNA. In order to produce cDNA, reverse transcriptase enzyme and other necessary components should be provided, especially template and primers. Primers are the short DNA sequences specifically designed for the amplification of the target sequences. Random primers and oligo dT primers are two common types of primers used in reverse transcription. Therefore, random primer or oligo dT primer are short DNA oligonucleotide sequences required for reverse transcriptase to initiate reverse transcription. Depending on the RNA template, the suitable primer can be selected out of these two primer types.

CONTENTS

1. Overview and Key Difference
2. What are Random Primers 
3. What is Oligo dT
4. Similarities Between Random Primers and Oligo dT
5. Side by Side Comparison – Random Primers vs Oligo dT in Tabular Form
6. Summary

What are Random Primers?

Random primer is a mixture of oligonucleotides representing all possible hexamer sequences. Primer sequence of random primer is 5´ – d (NNNNNN) –3´ N = G, A, T or C. Therefore, random primers can be used for the amplification of single-stranded DNA or RNA by DNA polymerase or reverse transcriptase, respectively. Besides, random primer mixture is capable of amplifying all regions of the RNA to generate cDNA. Thus, it produces various lengths of cDNA.

Most importantly, the random primer mixture does not show template specificity. It is incapable of distinguishing mRNA and other RNA species. And, it anneals with any species of RNA in the sample. However, random primer mixture is suitable for reverse transcription of RNAs without poly(A) tails, such as rRNA, tRNA, non-coding RNAs, small RNAs, prokaryotic mRNA etc., degraded RNA, and RNA with known secondary structures.

What is Oligo dT?

Oligo dT primer is a stretch of 12 – 18 deoxythymine (dT). The primer sequence can be represented as 5´-d (TTT TTT TTT TTT TTT TTT)-3. It is used for the production of cDNA from mRNA containing poly(A) tail. Basically, it acts as a primer for the reaction catalyzed by reverse transcriptase. During the reverse transcription, oligo dT primer anneals with the poly-adenylated tail found on the 3´ end of most eukaryotic mRNAs and starts the process.

Unlike random primer, oligo dT primer is not suitable for degraded RNA or RNA that lack poly(A) tails, including prokaryotic RNA and microRNA.

What are the Similarities Between Random Primers and Oligo dT?

• Random primer and oligo dT primer are two types of primers that are used in reverse transcription and making cDNA.
• Using the random primer and oligo dT primer together increases the sensitivity of cDNA synthesis.
• They are single-stranded short nucleotide sequences.

What is the Difference Between Random Primers and Oligo dT?

Random primer is a short oligonucleotide sequence composed of a random sequence. Meanwhile, oligo dT primer is a short strand composed of 12 to 18 deoxythymidines. So, this is the key difference between random primers and oligo dT. The sequence of random primer is 5´ – d (NNNNNN) –3´ N = G, A, T or C. Whereas, the sequence of oligo dT primer is 5´-d (TTT TTT TTT TTT TTT TTT)-3´. Therefore, we can consider this as another difference between random primers and oligo dT.

Moreover, random primers do not show template specificity, and they anneal with any species of RNA, while oligo dT primers show specificity for poly(A) tails of eukaryotic mRNA.

The below infographic summarizes the difference between random primers and oligo dT.

Summary – Random Primers vs Oligo dT

Random primer and oligo dT primer are two types of primers used in cDNA synthesis. Random primer consists of a mixture of oligonucleotides, representing all possible hexamer sequences, while oligo dT primer is a single-stranded sequence of 12 to 18 deoxythymidines. So, this is the key difference between random primers and oligo dT. Besides, oligo dT primers are useful for full-length reverse transcription of RNA with poly(A) tail, while random primers are useful for reverse transcription of most RNA species, including degraded RNA, RNA that lack poly(A) tails and RNA containing secondary structures. Therefore, the random primer does not show template specificity while oligo dT primer shows specificity towards eukaryotic mRNA containing poly(A) tail.

Reference:

1. “Reverse Transcription Setup: Thermo Fisher Scientific – US.” Reverse Transcription Setup | Thermo Fisher Scientific – US, Available here.

Image Courtesy:

1. “Primers RevComp” By Zephyris – Own work (CC BY-SA 3.0) via Commons Wikimedia
2. “RT PCR Model” By Lokeshthimmana – Own work (CC BY-SA 4.0) via Commons Wikimedia