Probe for closing ribosome RNA or globulin RNA in RNA library building process and application thereof
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A ribosome and globulin technology, applied in the field of probes for blocking ribosomal RNA or globulin RNA
Pending Publication Date: 2021-12-31
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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Therefore, there are still application limitations in using this method to remove ribosomal RNA and globulin RNA during RNA library construction
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Embodiment 1
[0064] Example 1: Inhibitory effect of different modification types of probes on target RNA reverse transcription
specific Embodiment approach
[0065] refer to figure 2 In this example, 5.8S-specific probes with different modifications (No. 1-5 in Table 1) were designed to inhibit the reverse transcription process of 5.8SrRNA. The specific implementation is as follows:
[0066] Table 2
[0067] components Dosage Total RNA 1μg 1 μM 5.8S modified probe (any one of No. 1-5 in Table 1) 1μL 1μM 5.8S rRNA RT primer 1μL 10mM dNTPs mix (with 0.5M KCl) 1μL Supplement DEPC H2O to 13μL
[0068] Mix and spin away. React at 75°C for 1 min and store at 4°C.
[0069] table 3
[0070]
[0071] Mix and spin away. React at 25°C for 10 minutes, at 42°C for 15 minutes, at 70°C for 5 minutes, and store at 4°C;
[0072] After diluting 1000 times, Hieff qPCR SYBR Green Master Mix (11200ES03) was used to quantify 5.8 ScDNA, and the quantitative primers are listed in Table 1. Quantitative results see image 3 .
[0073] From the quantitative results, it can be seen that peptide...
Embodiment 2
[0076] Example 2: The inhibitory effect of the distribution position of PNA in the probe on the reverse transcription of target RNA
[0077] In this example, 5.8S-specific probes (a mixture of sequences No. 6-8 in Table 1) were designed at different PNA modification positions to inhibit the reverse transcription process of 5.8S rRNA. The specific embodiment is with reference to embodiment 1, quantitative result sees Figure 4 .
[0078] It can be seen from the quantitative results that when the position of the PNA in the probe is close to the left end (ie, the 5' end) of the pairing region between the probe and the target RNA, the effect of hindering the reverse transcription of the target RNA is higher than that when the position of the PNA in the probe is close to the probe. The right end of the needle (i.e. the 3' end).
[0079] Table 5: Inhibition efficiency of target RNA reverse transcription by LNA distribution position in the probe
[0080] Distribution of ...
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Abstract
The invention provides a probe for blocking ribosome RNA or globulin RNA in the RNA library building process, the length of the probe is 35-60 nt, and the probe and the ribosome RNA or globulin RNA form a ternary blocking compound through complementary pairing. The rear half region of the probe is in strict complementary pairing with the target RNA, and 5-9 bases at the 5' end of the probe pairing region are replaced by modified bases; and the front half region of the probe is a pyrimidine base which is used for being embedded into a large groove of a DNA / RNA double strand to form a ternary complex. The 3'-OH of the probe is sealed by MGB for preventing extension of the probe as a primer; and meanwhile, the MGB can be embedded into a small groove of DNA / RNA double strands, so that the stability of the ternary complex is improved. The invention also discloses an application of the gene in rapid removal of ribosome RNA or globulin RNA in RNA library construction. The probe has the advantages of simplicity in operation, extremely short time consumption, high specificity, wide applicability, high removal efficiency, high gene detection number and the like, and is suitable for the fields of industrial automatic library building and rapid disease diagnosis.
Description
technical field [0001] The patent of the invention relates to a probe for blocking ribosomal RNA or globulin RNA in the process of building an RNA library, which belongs to the field of biotechnology. Background technique [0002] In recent years, with the rapid development of high-throughput sequencing technology, various RNA library construction and sequencing technologies have emerged and iteratively upgraded, greatly reducing the difficulty and cost of RNA library construction and sequencing, which has also made RNA high-throughput sequencing Technology has become an important research tool in fields such as life progression and disease diagnosis. However, the proportion of RNA species in samples from different sources is extremely heterogeneous. For example, ribosomal RNA (rRNA) accounts for about 90%-95% of the total RNA in normal cells, and globulin mRNA accounts for about 95% of the total RNA in blood. More than 76% of mRNA, the presence of these RNAs occupies most ...
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