IncRNA (long non-coding ribonucleic acid) expression difference-based isogonium identification method and application thereof

Abstract

The invention relates to the technical field of forensic medicine and molecular biology, and discloses an isomerism twin identification method based on long-chain non-coding RNA (lncRNA) expression difference and application of the isomerism twin identification method. In order to solve the problems that the existing DNA typing technology cannot distinguish the same-egg twin and the stability of epigenetic markers is poor, the lncRNA molecules differentially expressed between the same-egg twin are screened through high-throughput sequencing, and the real-time fluorescent quantitative PCR (qPCR) is used for verification. The key lncRNA marker MSTRG.73422.1 (LncR3) has a remarkable expression difference in all the 9 pairs of twentys, and the key lncRNA marker MSTRG.73422.1 (LncR3) has a remarkable expression difference. The method provided by the invention can still stably detect bloodstains preserved at room temperature for 180 days and 10 times of repeated freezing and thawing, and has high stability, high sensitivity and high specificity. The technical process is based on a qPCR platform, operation is easy and convenient, the method is suitable for a conventional forensic medicine laboratory, and the technical problem of identification of homozygous twin individuals is solved.

Description

Technical Field

[0001] This invention belongs to the fields of forensic medicine and molecular biology, specifically relating to a method for identifying identical twins based on differences in the expression of long non-coding RNAs (lncRNAs) and its application. Background Technology

[0002] In forensic practice, individual identification is a crucial step in case investigation and judicial proceedings. However, when the individuals involved are monozygotic twins (MZTs), traditional DNA typing techniques face fundamental challenges. Because monozygotic twins originate from the same fertilized egg, their genomic DNA sequences are almost identical, rendering short tandem repeat (STR) and single nucleotide polymorphism (SNP) analyses, based on DNA sequence differences, ineffective in distinguishing between the two. Although researchers have attempted to introduce copy number variation (CNV) or mitochondrial DNA heterogeneity as supplementary methods, these differences occur extremely rarely in real-world samples and have poor reproducibility, making them unreliable for routine identification. Currently, there is a lack of systematically screened and validated stable biomarkers, resulting in limited identification coverage and insufficient reproducibility, severely restricting their application in actual cases. Summary of the Invention

[0003] To address the shortcomings of existing technologies, such as their inability to effectively distinguish between identical twins with almost identical genome sequences, poor stability of epigenetic markers, low detection repeatability, and insufficient practical adaptability, the present invention aims to provide a method for identifying identical twins based on differences in the expression of long non-coding RNAs (lncRNAs).

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0005] This invention discloses a kit for identifying identical twins, characterized in that it contains reagents for detecting the expression level of lncRNA molecules; the lncRNA molecules are selected from one or more combinations thereof: MSTRG.30665.2, ENST00000414030, MSTRG.73422.1, MSTRG.87582.24, MSTRG.100816.5, MSTRG.54028.15, MSTRG.16169.7, MSTRG.98182.1, MSTRG.68271.29, MSTRG.63897.85, MSTRG.16294.1.

[0006] Furthermore, the kit contains specific primers for detecting lncRNA molecule expression levels.

[0007] Furthermore, the specific primer sequences for amplifying the UGT3A2 gene are shown in SEQ ID NO.1~SEQ ID NO.22.

[0008] Furthermore, the application of any of the above-described reagent kits in forensic individual identification or identification of identical twins.

[0009] The present invention also discloses a method for identifying identical twins based on differences in LncRNA expression, characterized in that the method uses the kit described in any of the above-mentioned claims to detect the LncRNA expression level of the sample.

[0010] Furthermore, the sample is tissue, blood, or cells.

[0011] Furthermore, the method described in any of the above can be applied to the identification of identical twins in forensic medicine.

[0012] The present invention also discloses a combination of lncRNA molecules for identifying identical twins, characterized in that the combination comprises MSTRG.73422.1, and one or two lncRNA molecules selected from MSTRG.30665.2 and ENST00000414030.

[0013] This invention introduces long non-coding RNAs (lncRNAs) as novel epigenetic markers and combines high-throughput sequencing with real-time quantitative PCR (qPCR) technology to systematically screen and validate a group of lncRNA molecules that are stably differentially expressed in identical twins. Among them, MSTRG.73422.1 (LncR3) showed significant differences in all 9 pairs of twins, demonstrating strong individual identification ability. More importantly, these lncRNA markers exhibit excellent stability under common forensic conditions: they remain stable in bloodstain samples stored at room temperature for 180 days, and their expression levels show no significant change after 10 repeated freeze-thaw cycles, completely overcoming the key shortcomings of existing epigenetic markers, such as easy failure in degraded samples and poor reproducibility. By constructing a "1+2+N" multi-lncRNA joint recognition system (ultimately a total of 9 lncRNAs with different distinguishing abilities: 1 is LncR3 (MSTRG.73422.1), which can distinguish all 9 pairs of twins; 2 are LncR1 (MSTRG.30665.2) and LncR2 (ENST00000414030) as secondary core markers, both of which can effectively distinguish 8 pairs of twins; N is other lncRNAs with supplementary recognition abilities introduced as extension modules according to the specific needs of the case, such as LncR11 (MSTRG.16294.1) and LncR4 (MSTRG.87582.24), to improve the overall recognition coverage and accuracy), the coverage and accuracy of individual identification were significantly improved, breaking through the bottleneck of the limited recognition ability of a single marker.

[0014] In summary, this invention effectively overcomes the fundamental defect of traditional DNA typing in distinguishing identical twins, and at the same time solves the shortcomings of existing epigenetic methods in terms of stability, sensitivity, standardization and practical adaptability, providing a new, stable, standardizable and applicable technical approach for forensic individual identification.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] 1. Achieving "full coverage" individual identification of identical twins for the first time. This invention is the first to discover that MSTRG.73422.1 (LncR3) shows significant and stable expression differences in all 9 pairs of identical twins, overcoming the shortcomings of low identification coverage in existing technologies and truly achieving universal differentiation of MZTs.

[0017] 2. The stability of the biomarkers is significantly superior to existing epigenetic methods. Compared with DNA methylation (which is susceptible to environmental interference and technically complex), the lncRNAs screened in this invention remain stable and detectable in bloodstains stored at room temperature for 180 days and after 10 repeated freeze-thaw cycles, significantly improving their applicability and reliability in commonly degraded samples in forensic medicine.

[0018] 3. The technical process is standardized and adaptable to existing forensic laboratory conditions. Compared to methylation detection, which requires sulfite conversion and has a complex process, this invention is based on a qPCR platform to extract RNA from peripheral blood or dried blood spots and perform expression analysis. It is simple to operate, highly reproducible, requires no additional equipment or special reagents, and is more suitable for widespread promotion and practical application.

[0019] 4. Applicable to twin samples of different ages and environmental backgrounds. This invention also detected significant differential expression in neonatal twins, indicating that its recognition ability is not limited by age or environmental exposure history, which is superior to existing technologies that partially rely on postnatal environmental differences, and has stronger population universality and developmental stage adaptability.

[0020] In summary, this invention not only achieves stable, efficient, and comprehensive differentiation of identical twins for the first time, but also significantly outperforms existing epigenetic methods in terms of marker stability, technical adaptability, and identification accuracy. It fills the technical gap in the identification of MZTs individuals in the field of forensic medicine and has outstanding practical value and prospects for promotion. Attached Figure Description

[0021] Figure 1 Venn diagram of differentially expressed lncRNAs in four pairs of identical twins.

[0022] Figure 2 The relative abundance levels of 11 candidate lncRNAs detected by qPCR in 4 pairs of sequenced twins. (A and B represent two individuals in a pair of identical twins. * indicates p-value < 0.05, paired t-test). qPCR validation results of the 11 lncRNAs in 4 pairs of twins.

[0023] Figure 3 The relative abundance levels of 11 candidate lncRNAs detected by qPCR in 5 pairs of non-sequencing twins. (A and B represent two individuals in a pair of identical twins. * indicates p-value < 0.05, paired t-test). qPCR validation results of the 11 lncRNAs in 5 pairs of validation twins.

[0024] Figure 4 The relative abundance and Cq values ​​of 9 candidate LncRNAs under different exposure durations. (* indicates p-value < 0.05, RM one-way ANOVA).

[0025] Figure 5 .9 relative quantities and Cq value changes of 9 candidate LncRNAs in different f / t cycles. (No statistical differences were found by one-way ANOVA of RM). Specific implementation manners

[0026] The present invention will be further described in detail below with specific embodiments. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies implemented based on the content of the present invention belong to the scope of the present invention.

[0027] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0028] I. Materials and methods.

[0029] 1. Main reagents and instruments.

[0030] RNA extraction: RNAiso Blood (TaKaRa, Code 9109); reverse transcription: PrimeScript RT kit (containing gDNA Eraser, TaKaRa RR047A); qPCR: TB Green Premix Ex Taq II (TaKaRa RR820A), ROX Reference Dye (50×); quantification platform: NanoDrop-2000 (Thermo Fisher); qPCR instrument: ABI 7500Fast Real-Time PCR System (Applied Biosystems); sequencing platform: Illumina NovaSeq 6000, PE150, completed by Novogene (Beijing); main consumables: RNA-free pipette tips and EP tubes (Axygen), 0.2 mL eight-strip tubes (Bio-Rad).

[0031] 2. Sample sources and ethics.

[0032] 9 pairs of Chinese identical twins (6 pairs of adults, 3 pairs of neonates) and 10 healthy unrelated volunteers, 5 mL (for adults) or 0.8 mL (for neonates) of peripheral blood was collected in EDTA anticoagulant tubes. The study was approved by the Ethics Committee of China Medical University (approval document 2024-Lenshen-080), and all subjects signed informed consent. The monozygotic twin relationship was verified by the SiFaSTRTM 23plex STR system, and the PI value > 10,000.

[0033] Of the nine Chinese identical twins mentioned above, four pairs served as the selection set. High-throughput library construction and sequencing were used to screen for 31 differentially expressed lncRNAs, which were then validated by PCR. Eleven of these lncRNAs, exhibiting high specificity and stable amplification, were selected. Five non-sequencing twin pairs were then added to form a validation set, totaling nine pairs, to test the 11 PCR-validated lncRNAs.

[0034] 3. RNA extraction and quality control.

[0035] Take 250 μL of fresh whole blood or one 0.5 cm tablet. 2 Dried blood spots were lysed, phase-separated, precipitated, and washed with 75% ethanol according to the RNAiso Blood instructions.

[0036] RNA precipitate was dissolved in 30 μL of DEPC water; NanoDrop assay concentration ≥20 ng / μL, A260 / 280=1.8-2.0, Agilent 2100 RIN≥6.5 is considered acceptable.

[0037] Qualified samples should be stored at −80℃ immediately and reverse transcribed within 4 hours.

[0038] 4. Library construction and lncRNA sequencing (LncRNA-seq).

[0039] Library construction, clustering, and sequencing.

[0040] The simplified workflow is as follows: First, 3′ and 5′ adapters were ligated to RNA, followed by reverse transcription to synthesize first-strand cDNA, and then PCR amplification and fragment screening. LncRNA libraries were constructed using PAGE gel electrophoresis and gel extraction. Finally, PCR products were purified using the AMPure XP system (BECKMAN COULTER Life Sciences, Indiana, USA), and library quality was assessed. Indexed samples were clustered using the cBot clustering system (Illumina, California, USA), followed by paired-end 150 bp (PE150) sequencing using the Illumina NovaSeq 6000 platform. A total of 173.21 Gb of clean data was obtained from long non-coding RNA analysis, with each sample achieving 15.64 Gb of clean data, yielding 32,992 lncRNAs.

[0041] After the 3'-5' linker is ligated, it is reverse transcribed into cDNA.

[0042] After 15 cycles of PCR enrichment, 300-600 bp fragments were excised from a 2% agarose gel.

[0043] Purification with AMPure XP magnetic beads, quantification with Qubit 3.0.

[0044] After cBot clusters are generated, NovaSeq 6000 PE150 sequencing is performed, with single-sample clean data ≥15 Gb and Q30 ≥90%.

[0045] 5. Differential lncRNA screening and functional annotation.

[0046] The raw data were quality controlled by FASTP and HISAT2 was used to GRCh38.

[0047] Quantitative analysis using StringTie (v2.1) and differential analysis using edgeR (|log2FC|≥0.58, FDR<0.05).

[0048] A total of 31 lncRNAs from four pairs of twins were differentially expressed in ≥3 pairs. Among them, 11 (LncR1-11) met the criteria of CV<2%, NTC Ct>35, and amplification efficiency of 90-110%, and were used for downstream validation.

[0049] 6. qPCR validation and stability testing.

[0050] 6.1 Reverse transcription.

[0051] 20μL system: RNA 400ng, gDNA Eraser 1μL, 5×gDNA Eraser Buffer 4μL, 42℃ for 2min; add 5×PrimeScript Buffer 4μL, RT Enzyme Mix I 1μL, 37℃ 15min→85℃ 5s.

[0052] 6.2 Primers and reactions.

[0053] qPCR system (20μL): cDNA 2μL, TB Green 10μL, primer final concentration 0.4μM, ROX 0.4μL; cycling: 95℃ 30s→95℃ 5s / 60℃ 34s×40, melting curve collected at 0.5℃ gradient.

[0054] The primer sequences are shown in Table 1 below.

[0055] Table 1 Primer sequences.

[0056] 6.3 Stability design.

[0057] Room temperature bloodstain group: 100 μL of blood was spread on quantitative filter paper and stored for 0, 3, 7, 14, 30, 90 and 180 days (n=3).

[0058] Freeze-thaw group: Liquid blood -80℃↔4℃ was circulated 1, 2, 3, 5, and 10 times (n=3).

[0059] With 2 −ΔΔCt The relative expression level was calculated using the method with 18S rRNA as the internal reference, and Ct≤35 was considered as detected.

[0060] II. Results.

[0061] 1. Differential expression profile.

[0062] Annotation and analysis of lncRNA sequencing data identified 12,547 expressed lncRNAs in four identical twin (MZT) pairs. Using |log2(fold change)|>1 and adjusted p-value <0.05 as screening criteria, 3330, 4323, 6118, and 4808 differentially expressed lncRNAs were identified in MZT-1, MZT-2, MZT-3, and MZT-4, respectively. Figure 1 Further analysis revealed that 31, 713, and 4013 DELncRNAs were shared in 4, 3, and 2 pairs of twins, respectively. Figure 1 By performing PCR pre-screening on 31 co-differentially expressed lncRNAs, 11 lncRNAs (LncR1-LncR11) with high specificity and stable amplification were obtained.

[0063] 2. Identify differentially expressed LncRNAs.

[0064] (1) qPCR detection of LncRNA in four pairs of sequencing twins.

[0065] To validate the lncRNA sequencing results and screen for differentially expressed lncRNAs with stable expression, qPCR analysis was performed on 11 lncRNAs that were differentially expressed in all four pairs of twins. Figure 2 The results showed that LncR1, LncR3, and LncR7 expression differed significantly among the four pairs of twins, consistent with sequencing results (FC>1.5 or <0.67 and P<0.05). LncR2, LncR4, LncR6, LncR8, LncR10, and LncR11 showed a trend consistent with sequencing in three pairs of twins; LncR5 and LncR9 showed consistency only in 1-2 pairs (Table 2).

[0066] Table 2. Expression profiles of 11 candidate long noncoding RNAs in four pairs of monozygotic twins (MZT-1–MZT-4): raw RNA sequencing counts, FPKM values ​​and significance parameters.

[0067] (2) Validation was performed using qPCR in five additional non-sequencing pairs of twins.

[0068] LncR3 expression was significantly different in five pairs of twins (FC>1.5 or <0.67 and P<0.05); LncR1, LncR2, and LncR11 expression was significantly different in four pairs of twins; LncR4, LncR7, and LncR8 expression was significantly different in three pairs of twins; LncR5, LncR6, LncR9, and Lnc10 expression was significantly different in only two pairs. Figure 3 ).

[0069] Stability and persistence of 3 candidate 11 LncRNAs.

[0070] (1) The effect of different exposure times.

[0071] Even after 180 days, nine lncRNAs could still be successfully detected (LncR5 and LncR7 were severely degraded), with Cq values ​​all below 35. Figure 4 Although the Cq value of LncR8 was slightly higher than 35 in some samples, its melting curve still showed effective amplification (Figure S1). The relative expression levels (RQ) of LncR2, LncR8, LncR9, and LncR10 did not change significantly within 180 days, indicating good stability of the bloodstain samples. Although the RQ of LncR3 and LncR6 showed a decreasing trend within 180 days, the difference was not statistically significant. The RQ of LncR1, LncR4, and LncR11 remained stable for the first 30 days, and then increased. The increase in LncR1 and LncR4 was not significant, while the increase in LncR11 was more significant, which may be related to the faster degradation rate of the internal reference gene, further confirming its stability.

[0072] (2) The effects of repeated freeze-thaw cycles.

[0073] The Cq and RQ values ​​of nine candidate LncRNAs were evaluated under different freeze-thaw cycles. Figure 5 All lncRNAs had Cq values ​​below 35, and the melting curves still showed effective amplification. RQ values ​​did not show significant differences, indicating that these lncRNAs have strong tolerance to repeated freeze-thaw cycles.

[0074] (3) Identification ability and applicability of candidate LncRNAs.

[0075] LncR3 exhibited the strongest distinguishing efficacy: it showed significant differential expression in all 9 pairs of identical twins, and remained detectable even after 180 days of exposure to room temperature and repeated freeze-thaw cycles, making it the most valuable for forensic practice and recommended as the primary indicator. LncR2 and LncR3 were comparable in distinguishing 8 pairs of twins, but LncR7 degraded significantly under the same conditions, resulting in poor field adaptability; if laboratory conditions are controllable, it can be combined with LncR2 as a secondary supplement. LncR11 could detect 7 pairs of differences and had excellent stability and persistence, possessing the potential to be a "sub-first choice" indicator, suitable for long-term samples. The remaining LncRNAs had a distinguishing number of ≤6 pairs, with LncR5 showing significant degradation, directly excluding it from forensic applications.

[0076] in conclusion.

[0077] The above implementation steps demonstrate that the lncRNA-qPCR protocol provided by this invention has the characteristics of high specificity, high stability, and high sensitivity. It can be repeatedly and standardizedly applied to routine forensic laboratories to achieve effective differentiation of identical twins.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kit for identifying identical twins, characterized in that, It contains reagents for detecting the expression level of lncRNA molecules; the lncRNA molecules are selected from one or more combinations thereof: MSTRG.30665.2, ENST00000414030, MSTRG.73422.1, MSTRG.87582.24, MSTRG.100816.5, MSTRG.54028.15, MSTRG.16169.7, MSTRG.98182.1, MSTRG.68271.29, MSTRG.63897.85, MSTRG.16294.

1.

2. The reagent kit according to claim 1, characterized in that, The kit contains specific primers for detecting lncRNA molecule expression levels.

3. The reagent kit according to claim 2, characterized in that, The specific primer sequences for amplifying the UGT3A2 gene are shown in SEQ ID NO.1 to SEQ ID NO.

22.

4. The application of the kit according to any one of claims 1-3 in forensic individual identification or identification of identical twins.

5. A method for identifying identical twins based on differences in LncRNA expression, characterized in that, The method uses the kit described in any one of claims 1 to 3 to detect the expression level of LncRNA in the sample.

6. The method according to claim 5, characterized in that, The sample may be tissue, blood, or cells.

7. The application of the method according to any one of claims 5 to 6 in forensic identification of identical twins.

8. A lncRNA molecular combination for identifying identical twins, characterized in that, The combination comprises MSTRG.73422.1, and one or two lncRNA molecules selected from MSTRG.30665.2 and ENST00000414030.

Images