A Remarkable Breakthrough in Strand-specific RNA Library Preparation

Have you ever faced doubts while constructing an RNA library? Concerns about the toxicity of certain reagents and the need to work in a light-free environment can be quite perplexing, particularly when dealing with numerous samples. Actinomycin D has often been the source of these concerns, as it is known for its toxicity and light sensitivity. But why is actinomycin D included in library kits?

Recent findings have revealed that actinomycin D possesses the ability to inhibit transcription. It achieves this by binding to DNA at the transcription initiation complex, thereby preventing the elongation of RNA chains by RNA polymerase. Essentially, actinomycin D interferes with polymerase activity, hindering the simultaneous synthesis of the second strand during the cDNA synthesis process. This allows for the use of dUTP instead of dTTP in two-strand synthesis, leading to enhanced chain specificity in RNA library construction.

The incorporation of actinomycin D in RNA library constructing kits has undeniably improved the construction of strand-specific libraries, significantly enhancing chain specificity. This innovation has streamlined the experimental process, simplifying it for researchers. Moreover, it has substantially improved chain specificity, a pivotal aspect of RNA-related scientific inquiries.

Figure 1: Comparative Test Results by NEB Company: Impact of Actinomycin D. The data depicted in the graph reveals a remarkable outcome. Over 90% of the reads were aligned with the anticipated positive-sense strand, demonstrating the excellent chain specificity of the sequence library that includes actinomycin D. (From NEB website)

However, actinomycin D does have its drawbacks: it exhibits toxicity and requires protection from light. In today's landscape of increasing demand for premixed and plate library constructing kits, the necessity to shield against light poses limitations on plate kit advancements.

Fortunately, there is no longer a need to be concerned about actinomycin D. The Yeasen ZymeEditor platform has introduced a groundbreaking MMLV enzyme mutant that replaces the function of actinomycin D. This new Kit is odorless, non-toxic, and no need to avoid light. It offers superior chain specificity, eliminating concerns related to health and light sensitivity.

Figure 2: Engineering of MMLV to identify MMLV mutants which could contribute to Standed RNA-seq 

	12301ES	12308ES	12310ES	12340ES
Actinomycin D	√	√	√	×
MGI	×	√	√	√
Illumina	√	√	√	√
Premix	×	×	√	√
Component quantity	10	10	6	5
Release Year	2017	2021	2022	2023

Table 1： Distinguishing Features of Yeasen's RNA Lib Prep Kits

Figure 3. When employing the kit containing actinomycin D alongside the new kit lacking actinomycin D, the yields were comparable for both kits at RNA inputs of 10 ng and 1 μg. However, at an RNA input of 4 μg, the new kit without actinomycin D exhibited higher yields.

Figure 4. The utilization of the new kit, featuring an MMLV mutant without the addition of Actinomycin D, results in superior strand specificity compared to the kit that incorporates Actinomycin D. This distinction becomes more pronounced, particularly at lower RNA inputs of 10 ng.

Yeasen, dedicated to enhancing the customer experience, continues to break new ground in the realm of RNA library kits.

 

Product recommendation

 

Name	Cat#	Size
Hieff NGS ® EvoMax RNA Library Prep Kit（Premixed version）(actinomycin D Free)	12340ES24/96	24 T/ 96 T
Hieff NGS® Ultima Dual-mode RNA Library Prep Kit(Premixed version) (With actinomycin D)	12310ES24/96	24 T/ 96 T
Hieff NGS™ Ultima Dual-mode mRNA Library Prep Kit	12308ES24/96	24 T/ 96 T
Hieff NGS® mRNA Isolation Master Kit V2	12629ES24/96	24 T/ 96 T
Hieff NGS MaxUp Human rRNA Depletion Kit(rRNA & ITS/ETS,H/M/R)	12257ES24/96	24 T/ 96 T
Hieff NGS® MaxUp rRNA Depletion Kit(Plant)	12254ES24/96	24 T/ 96 T
Hieff NGS® MagSP rRNA Removal Kit (Bacteria(G- and G+)) with purification beads	12264ES24/96	24 T/ 96 T