Reverse Transcription

Overview – Reverse Transcription

• Function
  • convert RNA to DNA
  • performed by reverse transcriptase
    • has high error rate and cannot proofread
    • three activities
      • RNA-dependent DNA polymerase
      • RNase
      • DNA-dependent DNA polymerase
• Clinical relevance
  • present in retroviruses
    • e.g. HIV
      • some HIV drugs inhibit reverse transcriptase
        • AZT
          • enters cells and modified by addition of ATP
          • functions as a nucleoside analog
          • terminates replication
  • present in telomerase
    • see Telomere topic 
  • used in RT-PCR
• see PCR topic 

Introduction

Reverse transcription is a molecular biology technique that allows for the conversion of RNA into complementary DNA (cDNA) using reverse transcriptase enzyme. This process is the reverse of the typical flow of genetic information from DNA to RNA during transcription. Reverse transcription is important in many biological applications, such as gene expression analysis, cloning, and viral studies.

The reverse transcriptase enzyme used in this process is derived from retroviruses and is capable of synthesizing a complementary DNA strand from an RNA template. This enzyme has a high processivity and fidelity, making it a reliable tool for cDNA synthesis.

Types

There are several types of reverse transcription methods that can be used depending on the application and the type of RNA being analyzed. Here are some of the common types of reverse transcription:

1. First-strand cDNA synthesis: This method involves the use of an oligo(dT) primer that hybridizes to the poly(A) tail of mRNA. Reverse transcriptase synthesizes the first strand of cDNA using the primer as a starting point.
2. Random priming: In this method, random hexamers are used as primers for cDNA synthesis. These primers hybridize to random locations along the RNA template, resulting in the synthesis of a representative pool of cDNA.
3. Quantitative reverse transcription PCR (qRT-PCR): This method involves the use of reverse transcription to convert RNA to cDNA, followed by PCR amplification of the cDNA using primers specific to the target gene. The amount of cDNA produced is proportional to the amount of RNA present in the sample, allowing for quantitative analysis of gene expression.
4. Multiplex reverse transcription PCR: This method involves the simultaneous reverse transcription of multiple RNA targets using multiple primers, followed by PCR amplification of the cDNA. This approach allows for the analysis of multiple RNA targets in a single reaction.
5. Strand-specific reverse transcription: This method involves the use of primers that anneal to specific RNA strands, allowing for the synthesis of cDNA from either the sense or antisense strand of RNA. This approach can be used to study the transcriptional regulation of genes and the expression of non-coding RNAs.

Function

Reverse transcription has several important functions in molecular biology:

1. Gene expression analysis: Reverse transcription is commonly used to measure gene expression levels by converting mRNA into cDNA, which can be analyzed by quantitative PCR, microarray analysis, or RNA sequencing.
2. Cloning: Reverse transcription can be used to clone RNA molecules by converting them into cDNA, which can then be amplified by PCR and inserted into a plasmid vector for expression in bacteria or other organisms.
3. Viral studies: Reverse transcription is an essential step in the replication cycle of retroviruses, such as HIV. The reverse transcriptase enzyme converts the RNA genome of the virus into DNA, which can then be integrated into the host cell genome.
4. Identification of novel RNA species: Reverse transcription can be used to identify novel RNA species, such as non-coding RNAs or alternative splicing isoforms, by converting them into cDNA for analysis.

Complications

1. RNA quality: The quality and integrity of RNA can significantly impact the success of reverse transcription. Degraded or fragmented RNA may not yield reliable cDNA, resulting in inaccurate downstream analysis.
2. RNA secondary structure: RNA can form complex secondary structures that can interfere with the binding of reverse transcriptase to the template, resulting in incomplete or inaccurate cDNA synthesis.
3. Inhibitory substances: Some substances, such as salts, detergents, or organic solvents, can inhibit reverse transcriptase activity, resulting in incomplete or inaccurate cDNA synthesis.
4. Incomplete reverse transcription: Reverse transcriptase can occasionally stop or pause during cDNA synthesis, resulting in incomplete cDNA molecules. This can affect downstream analysis and result in false negatives or inaccurate results.
5. Primer specificity: The specificity of primers used in reverse transcription can impact the accuracy of cDNA synthesis. Specific primers can ensure that only the RNA of interest is converted into cDNA, while non-specific primers can result in the synthesis of unwanted cDNA.
6. Contamination: Contamination with DNA or other substances can result in inaccurate results or false positives during downstream analysis.

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