CN-121975920-A - RNA abnormal splicing verification method and kit

Abstract

The invention relates to the technical field of molecular biology and genetic diagnosis, and particularly discloses an RNA abnormal splicing verification method and a kit, wherein the method comprises the steps of extracting whole blood RNA and reversely transcribing the whole blood RNA into cDNA; the cDNA is used as a template, the outside primer pair shown as SEQ ID NO.2 and SEQ ID NO.3 is used for carrying out first round PCR amplification to cover the 17 th to 26 th exons of the OTOF gene, the amplified product is subjected to preliminary judgment by electrophoresis, the second round of nested PCR amplification to cover the 20 th to 23 th exons is carried out by using the inside primer pair shown as SEQ ID NO.5 and SEQ ID NO.6 after the product is recovered, and finally the second round of amplified product is sequenced and the wild type sequence is compared to determine the abnormal mode. The kit comprises the primer pair. The invention is directly based on blood samples, has simple and convenient operation, avoids genome DNA pollution through the design of a trans-exon primer, and can accurately and specifically reveal the influence of the mutation on RNA splicing.

Inventors

• LIU ZEHUA
• JIANG XIN
• WANG YIN
• ZHU YUN
• Tang Shuainan

Assignees

• 南通中科医学检验实验室有限公司

Dates

Publication Date

20260505

Application Date

20251231

Claims (5)

1. 1. The method for verifying abnormal splicing of RNA is characterized by comprising the following steps: s1, extracting total RNA of whole blood of a person to be tested, and reversely transcribing the total RNA into cDNA; S2, taking the cDNA as a template, and performing first-round PCR amplification by using an outer primer pair OTOF-F2 and OTOF-R2, wherein the nucleotide sequence of the OTOF-F2 is shown as SEQ ID NO.2, and the nucleotide sequence of the OTOF-R2 is shown as SEQ ID NO. 3; s3, agarose gel electrophoresis is carried out on the amplified product in the step S2, and whether splicing is abnormal or not is primarily judged according to the size of the product fragment; S4, recovering an amplification product of the step S2, taking the amplification product as a template, and performing second-round nested PCR amplification by using an inner primer pair OTOF-E22-F2 and OTOF-E22-R2, wherein the nucleotide sequence of the OTOF-E22-F2 is shown as SEQ ID NO.5, and the nucleotide sequence of the OTOF-E22-R2 is shown as SEQ ID NO. 6; S5, sequencing and analyzing the amplified product in the step S4, and comparing the amplified product with a wild type sequence to determine whether the mutation of the OTOF gene c.2676G > A causes RNA splicing abnormality and a specific mode thereof.
2. 2. The method for verifying abnormal RNA splicing according to claim 1, wherein the target region amplified by the first round of PCR in step S2 covers exons 17 to 26 of the OTOF gene, and the theoretical amplification length is 1058bp.
3. 3. The method for verifying abnormal RNA splicing according to claim 1, wherein the target region amplified by the second round of nested PCR in step S4 covers exons 20 to 23 of the OTOF gene, and the theoretical amplification length is 384bp.
4. 4. A kit for verifying abnormal splicing of RNA is characterized by comprising the following components: The nucleotide sequence of the outer forward amplification primer OTOF-F2 is shown as SEQ ID NO. 2; the nucleotide sequence of the outside reverse amplification primer OTOF-R2 is shown as SEQ ID NO. 3; The nucleotide sequence of the inside forward amplification primer OTOF-E22-F2 is shown as SEQ ID NO. 5; The nucleotide sequence of the inside reverse amplification primer OTOF-E22-R2 is shown as SEQ ID NO. 6.
5. 5. A kit for validating aberrant splicing of RNA as defined in claim 4, wherein the kit further comprises one or more of reagents for reverse transcription, PCR amplification reagents, gel electrophoresis reagents, and/or sequencing reagents.

Description

RNA abnormal splicing verification method and kit Technical Field The invention relates to the technical field of molecular biology and genetic diagnosis, in particular to an RNA abnormal splicing verification method and a kit. Background RNA splicing is a key step in eukaryotic gene expression and refers to the process of removing introns (non-coding sequences) from primary RNA transcripts (e.g., hnrnas) produced by DNA transcription and ligating exons (coding sequences) into mature mRNA. This process is catalyzed by spliceosomes (complexes consisting of micronuclear RNA and protein) and effects intron excision and exon splicing by two transesterification reactions. RNA splicing ensures the correct coding information transmission of mRNA, and enables a single gene to generate various protein products through alternative splicing mechanisms, thereby having important significance for the development, functional regulation and evolution of organisms. RNA splicing abnormality may cause organ dysfunction and various diseases, and is one of the important causes of human genetic diseases. Mutations in both exons and non-coding sequences may cause splice abnormalities, and the reason for the mutation site affecting splicing may be activation of a hidden splice recognition site, formation of a new splice site, or the mutation site's splice regulatory sequence affecting target protein binding indirectly affecting splicing. In general, mRNA splicing abnormalities caused by mutation mostly lead to exon skipping, partial deletion of exons, deep intronic fragment retention, intronic fragment retention near boundaries, and the like. It is estimated that pathogenic splice abnormalities account for 10-50% of all pathogenic mutations, and that pathogenic mutations on or in introns (100 bp away from the intron/exon boundary) account for approximately 25%. The specific influence of the mutation site of the clear splicing region on RNA splicing is important to the evaluation of mutation pathogenicity, assists the further clinical diagnosis, assists the clinician in formulating clinical treatment strategies or provides basis for assisting reproduction. Common experimental techniques include direct analysis of RNA and/or cDNA derivatives, and in vitro minigene techniques, wherein biopsy tissue of a patient or animal model is more convincing to directly use as a subject, but the expression of genes, types of samples that can be collected, and types of samples that are more suitable for the literature report selection are used as experimental materials. The OTOF gene is located on chromosome 2 p23.3, and the gene has a total length of 101554 bases and 48 exons. The sensor is responsible for encoding otoferlin, which is calcium ion, contains 1997 amino acids, has 6 calcium ion binding C2 domains and is mainly involved in fusion and neurotransmitter release of synaptic vesicles related to inner hair cells and calcium ions, so that auditory neurons are activated. The NCBI and GENECARDS database are used for inquiring, the OTOF gene is expressed in blood, so that the in vitro verification experiment of mRNA abnormal splicing is carried out based on a blood sample, RNA is extracted from the blood sample, inverted into cDNA, a plurality of exons/introns close to a mutation site are amplified by a designed specific primer, and the influence of the mutation on mRNA splicing (such as exon deletion, intron retention and the like) is confirmed by utilizing electrophoresis and sequencing technology. The OTOF gene c.2676g > a mutation is located at the last nucleotide position of exon 22, which position mutation may affect the splice donor site, resulting in exon skipping or partial deletion, as predicted by bioinformatics. However, the actual effect of such 'boundary mutations' located near exon-intron boundaries on splicing is difficult to predict accurately by bioinformatics tools and must be verified experimentally. Currently, experimental methods to verify such mutations mainly include direct analysis based on patient tissue RNA and in vitro mimetic analysis based on Minigene or CRISPR/Cas 9. For the OTOF gene, because of its expression in peripheral blood, RNA can theoretically be obtained directly from the patient's blood for analysis, which is more straightforward and simple than in vitro simulation methods that require the construction of complex plasmids or cell lines. However, how to design efficient, specific amplification primers, how to clearly and accurately reveal the specific effect of the c.2676g > a mutation on splicing by reliable experimental procedures (especially for low abundance transcripts that may be present in blood samples), and no published, efficient standardized protocol is currently available. Therefore, the development of a direct detection method and a kit special for verifying the C.2676G > A mutation splicing effect of the OTOF gene has simple and convenient operation and accurate results, and has important