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New Arrival | So easy! Easy blocking of rRNA in 8-minute one-step operation (Ⅰ)

View: 284 / Time: 2024-08-27
01 Background
RNA sequencing (RNA-Seq) has become an indispensable and effective tool in basic biological research and disease diagnosis. It can not only detect the difference in the expression of all genes under different conditions, but also reveal the diversity of RNA structures, such as alternative splicing, gene fusions and single nucleotide  mutations.
Ribosomal RNA (rRNA) typically accounts for more than 80% of the total RNA in normal cells/tissues. The abundance of rRNAs can occupy most of the capacity and resources of sequencing, thus reducing the efficiency of detecting the target transcriptome information. Therefore, the removal of rRNA effects is a crucial step in RNA-Seq, contributing to improve the accuracy, efficiency and economics of studies, especially for the detection of low-abundance transcripts, ensuring that sequencing resources are more focused on the RNA molecules of interest.
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Figure 1. Distribution of RNA in mammalian cells.

02 RNA enrichment
The methods for mitigating the influence of rRNA removal in RNA-Seq applications can be divided into forward enrichment methods (such as Oligo dT magnetic bead enrichment of mRNA) and reverse enrichment methods (removal of rRNA).
2.1 mRNA enrichment by Oligo dT magnetic beads
In eukaryotes, most mRNA possess a ploy (A) tail, making it feasible to acquire transcriptome information using Oligo dT magnetic beads enrichment methods. While this method is relatively cost-effective, it also have some limitations:
①It is only applicable to eukaryotes, but not to prokaryotic RNA without ploy (A) tail;
②It is only applicable for RNA samples with good integrity, and mRNA with fragmented ploy (A) tail caused by degradation cannot be enriched;
③It only enriches mRNA and cannot enrich non-poly (A) tail-containing RNAs such as lncRNA/circRNA, significantly reducing the diversity of RNA-Seq;
④The enrichment efficiency depends on the length of poly (A) tail, resulting in the preference of RNA library preparation.
2.2 rRNA removal
The rRNA removal solution can be subdivided into magnetic bead-based method and enzymatic digestion method.
The magnetic bead-based method typically achieve rRNA removal through the following steps: Specific sequence of biotin-modified probes a bind to rRNA to form heterozygous chains; Streptavidin magnetic beads bind to hybrid chains containing rRNA, allowing for their capture on the surface of magnetic beads. Subsequent steps such as supernatant transfer are performed to remove rRNA.
Enzymatic digestion methods generally employ RNase H enzyme to digest rRNA:Specific sequence of DNA probes binds to rRNA to form heterozygous chains; RNase H enzyme hydrolyzes the rRNA in the heterozygous chain to single nucleotide or oligonucleotide. DNase I is added to digest the DNA probe.
While both of these rRNA removal methods have a wider scope of application, and can also remove rRNA well for partially degraded low-quality RNA samples, but their drawbacks are also obvious:
①High cost;
②Tedious operation involving approximately 20 pipetting steps;
③Time-consuming, requiring about 2 hr for rRNA removal.
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Figure 2. Comparison of different RNA enrichment methods

03 rRNA Fast Blocking Comprehensive Solution
Nanodigmbio has launched rRNA Fast Blocking Comprehensive Solution, aiming to achieve cost-effective and straightforward rRNA blocking. This solution requires only a single-step operation to rapidly and efficiently block rRNA, saving substantial time and effort. The core product, rRNA Blocking Reagent: NadPrep rRNA Blocking Reagent (Human), contains high-affinity oligonucleotides that bind to human rRNA，preventing its reverse transcription and significantly reducing the proportion of rRNA in sequencing libraries.
3.1 Solution principle and process
Before total RNA reverse transcription, rRNA Blocking Reagent (Human) preferentially binds to rRNA without affecting the binding of random primers and Oligo dT to RNA. During the reverse transcription process, rRNA Blocking Reagent can prevent the reverse transcription of rRNA, while non- rRNA can proceed smoothly, so as to achieve the effect of blocking.
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Figure 3. Blocking principle of rRNA Blocking Reagent (Human)

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Figure 4. Full process of rRNA Fast Blocking Comprehensive Solution (synthesis of double-stranded DNA)
3.2 Solution Features

rRNA Fast Blocking Comprehensive Solution, using NadPrep rRNA Blocking Reagent (Human) coupled with the NadPrep Total RNA-To-DNA Module, seamlessly connects to the NadPrep DNA Library Preparation Kit-series, enabling the rapid blocking of rRNA in total RNA. The Blocking Reagent is simple to use, requiring only a single pipetting step to add the rRNA Blocking Reagent (Human) to the reaction system. Following high-temperature fragmentation, an 8-minute gradient cooling reaction quickly blocks the rRNA in total RNA.

①Fast and convenient operation;

②Compatible with RNA samples of different quality;
③rRNA blocking only takes 8 min, and the whole process of double-stranded DNA synthesis takes 2.5hr;
④Effective enrichment of low abundance RNA, reducing sequencing costs;
⑤Suitable for third-party RNA library preparation kits.
3.3 Solution Performance
Compatible with Different Input Amounts for Various Sample Types
rRNA Fast Blocking Comprehensive Solution was applied to cell line RNA samples with high integrity, as well as A-grade and B-grade FFPE-derived RNA samples, with input amounts ranging from 50 ng to 500 ng. The results consistently showed that, for the same sample type, the blocking efficiency of rRNA in total RNA remained consistent across different input amounts. Furthermore, the blocking of rRNA did not affect the efficiency of RNA or the uniformity of transcript coverage.
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Figure 5. Performance of the NadPrep rRNA Fast Blocking Comprehensive Solution for different sample types and input amounts. A. rRNA ratio; B. No. of genes detected; C. Transcript coverage.


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