A New Era of RNA Library Prep: The World’s First Pre-mixed Kit Without Toxic Actinomycin D

In molecular biology, even the smallest breakthroughs can dramatically expand our understanding of life. When it comes to RNA library preparation, efficiency and accuracy are critical. Researchers handling hundreds or even thousands of samples often face significant challenges: time-consuming workflows, risks of contamination, and the constant pressure of meeting deadlines.

Traditionally, RNA library preparation involves multiple labor-intensive steps—fragmentation, first-strand synthesis, second-strand synthesis, adapter ligation, and library amplification. Each stage adds complexity, consumes time, and increases the risk of errors or contamination.

Today, however, the transition from manual to automated workflows is reshaping next-generation sequencing (NGS). The introduction of pre-mixed RNA library prep kits and plate-based pre-dispensed reagents, combined with automated library prep instruments, is streamlining processes like never before. Researchers can now enjoy faster, safer, and more consistent RNA library preparation.

Breaking Free from Actinomycin D

One of the central challenges in strand-specific RNA library prep has been dependence on actinomycin D. This molecule binds into the minor groove of DNA and forms a complex that blocks reverse transcriptase from synthesizing double-stranded cDNA during first-strand synthesis. Strand specificity is then achieved through the incorporation of dUTP during second-strand synthesis, followed by UDG-mediated digestion.

However, actinomycin D comes with significant drawbacks: it is toxic, requires light-protected storage, and introduces additional handling risks.

Yeasen has solved this problem through protein engineering. After extensive screening and evaluation, Yeasen developed a modified DNA polymerase variant with enhanced RNA-dependent activity. This breakthrough allows the enzyme to preferentially bind RNA templates during reverse transcription, eliminating the need for actinomycin D. The result: high-quality strand-specific RNA libraries without the toxicity and instability of actinomycin D.

Traditional vs. Yeasen Pre-mixed Workflow

Feature	Traditional Actinomycin D–Based Workflow	Yeasen Pre-mixed Kit (No Actinomycin D)
Strand Specificity	Achieved via actinomycin D + dUTP/UDG digestion	Achieved via engineered RNA-dependent polymerase
Safety	Uses toxic actinomycin D; requires light-protected handling	No toxic reagents; safer for researchers
Workflow Complexity	Multiple pipetting steps; higher contamination risk	Pre-mixed format; fewer steps, lower contamination risk
Storage & Handling	Actinomycin D requires special conditions	Standard laboratory storage
Automation Compatibility	Limited	Fully compatible with automated platforms(tube&plate-packing)
Scalability	Labor-intensive, less suited for high-throughput	Ideal for large-scale, high-throughput projects

 

Advantages of Yeasen’s Pre-mixed RNA Library Prep Kit

• High Strand Specificity – Advanced reverse transcriptase delivers highly strand-specific libraries.    
• Free of Toxic Actinomycin D – The only kit worldwide with a safe, light-stable formulation.
• Streamlined Workflow – Just 5 tubes with all components pre-mixed for ease of use.
• Fast Library Prep – cDNA synthesis, end repair, and A-tailing combined in one step, saving up to 2 hours.
• Automation - Friendly – Available in plate or tube packaging; ready-to-use format for high-throughput workflows.

1. Fast Workflow

 2. High-Yield, Flexible RNA Library Preparation

Figure 1. Comparison of RNA Library Yields.

(A, B) Strand-specific mRNA libraries from human, wheat, and mouse RNA (200 ng, 500 ng) using the Premix RNA Library Prep Kit (Cat#12340) with mRNA Isolation Master Kit (Cat#12629) showed higher yield than Supplier N*. (A) Qubit quantification. (B) qPCR quantification.

(C) Strand-specific LncRNA libraries from mouse RNA (200 ng, 500 ng) using the Premix RNA Library Prep Kit (Cat#12340) with rRNA Depletion Kit (Human/Mouse/Rat, Cat#12726) showed higher yield than Supplier K* (qPCR).

(D) Strand-specific LncRNA libraries from plant RNA (Arabidopsis, wheat, soybean leaves, 200 ng, 500 ng) using the Premix RNA Library Prep Kit with rRNA Depletion Kit (Plant, Cat#12254) showed higher yield than Supplier N* (qPCR).

3. Higher Strand Specificity, More Transcripts         

(A). Higher Strand Specificity

(B). Transcript Composition Analysis

                           

Figure 2. Comparison of mRNA Library Strand Specificity and Transcript Composition.

(A) Human mRNA libraries prepared from 200 ng and 500 ng input using 12340 demonstrated strand specificity of 99.4% and 99.3%, respectively, compared with 99.0% and 99.2% for Supplier N*.

(B) Transcript composition analysis showed that 12340 libraries consistently detected more transcripts. With 200 ng input, 12340 detected 89.3% of coding + UTR transcripts versus 84.9% for Supplier N*. At 500 ng input, 12340 detected 89.6% compared with 85.1% for Supplier N*.

4. Automation-Friendly

(A). Consistent Library Yield

(B). High Correlation in Gene Expression

                 

Figure 3. Consistency and Correlation Analysis.

(A) RNA standard samples (D5, D6, F7, M8; 3 replicates each) were processed using the NGS Premix RNA Library Prep Kit with NGS mRNA Isolation Master Kit (Cat#12629) on both automated and manual workflows. Comparable, highly uniform library yields were obtained across all samples.

(B) Gene expression fold-change analysis against the RNA standard dataset demonstrated strong concordance, with Pearson r² > 0.94. 

5. Strong Stability

(A).Accelerated stability

(B). Real-Time Stability

 Figure 4. Stability Evaluation of the Strand-Specific RNA Library Prep Kit.

(A) Accelerated stability was tested at 4 °C and 25 °C, with −20 °C storage as the control.

(B) Real-time stability was monitored for up to 9 months under both single-tube and standard packaging formats.

Category		Name	Cat. No.	Size
RNA Lib Prep	Dual-mode(Strand specific & Non Strand specific)	Hieff NGS™ Ultima Dual-mode RNA Library Prep Kit	12308ES24/96	24 T/96 T
	Premix version	Hieff NGS™ EvoMax RNA Library Prep Kit (Strand-specific)	12340ES24/96
		Hieff NGS™ EvoMax RNA Library Prep Kit（dNTP）	12341ES24/96
mRNA isolation	Eukaryotic mRNA	Hieff NGS™ mRNA Isolation Master Kit V2	12629ES	24 T/96 T
rRNA depletion	Human/Mouse/Rat	Hieff NGS™ MaxUp Human/Mouse/Rat rRNA Depletion Kit(rRNA ITS/ETS) 2.0	12726ES
	Plant	Hieff NGS™ MaxUp rRNA Depletion Kit (Plant)	12254ES
Beads	-	Hieff NGS™ RNA Cleaner	12602ES	1/5/60 mL