Reverse transcription (RT) is a fundamental process in RNA analysis, converting RNA into complementary DNA (cDNA). This step is essential for various techniques, including RNA sequencing (RNA-seq), which researchers use to study gene expression. Ensuring the effectiveness of this initial stage is vital, especially when dealing with small amounts of RNA.
Many factors can influence the efficiency of reverse transcription, such as the quality of the input material and the specific conditions of the reaction. However, one aspect that hasn’t been thoroughly explored is the length of the primers used during RT. Primers are short sequences of nucleotides that initiate the synthesis of cDNA from RNA. Understanding how the length of these primers affects the process can provide valuable insights and improvements in RNA analysis methods.
To investigate this, scientists at Friedrich Schiller University created RNA-seq libraries using random RT primers of different lengths: 6, 12, 18, or 24 nucleotides. Their findings were surprising. The 18-nucleotide primer (18mer) demonstrated superior efficiency in detecting transcripts compared to the commonly used 6-nucleotide primer (6mer). This was particularly true for detecting longer RNA transcripts in complex human tissue samples.
Rationale and experimental design of the study
A The pie chart visualizes the number of publications using random 6mers compared to the publications describing the use of random primers of other lengths between 4 and 24 nucleotides. B Schematic of the experimental workflow from RNA input to cDNA synthesis with random primers of different length in technical triplicates, cDNA pre-amplification, and sequencing library generation.
The results highlight the significant impact of primer length on the efficiency of reverse transcription. The superior performance of the 18mer primer suggests that optimizing primer length can enhance the detection of RNA transcripts. This improvement can benefit various applications, from basic research, where understanding gene expression is crucial, to clinical diagnostics, where accurate RNA detection can inform disease diagnosis and treatment.
This study underscores the importance of primer length in reverse transcription efficiency. By using longer primers, such as the 18mer, researchers can achieve better results in RNA analysis, paving the way for advancements in both research and clinical fields.
Micheel J, Safrastyan A, Aron F, Wollny D. (2024) Exploring the impact of primer length on efficient gene detection via high-throughput sequencing. Nat Commun 15(1):5858. [article]