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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 20, 2024 |
| Title |
Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [RNA-seq] |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications.
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| Overall design |
RNA sequencing. Total RNA was extracted using the Total RNA Extractor (Trizol) kit (B511311, Sangon, China) according to the manufacturer’s protocol, and treated with RNase-free DNase I to remove genomic DNA contamination. RNA integrity was evaluated with a 1.0% agarose gel. Thereafter, the quality and quantity of RNA were assessed using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and a Qubit® 2.0 Flurometer (Invitrogen). The high-quality RNA samples were subsequently submitted to the Sangon Biotech (Shanghai) Co., Ltd. for library preparation and sequencing. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina® following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in VAHTSTM First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs was converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3’ ends of DNA fragments, the adaptor was ligated to prepare for the library. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3-μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the NovaSeq sequencers (Illumina, San Diego, CA) Gene expression values of the transcripts were computed by StringTie (version 1.3.3b). Principal Component Analysis (PCA) and Principal co-ordinates analysis (PCoA) were performed to reflect the distance and difference between samples. The TPM (Transcripts Per Million), eliminates the influence of gene lengths and sequencing discrepancies to enable direct comparison of gene expression between samples.
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| Contributor(s) |
Xu S |
| Citation(s) |
38972974 |
| BioProject |
PRJNA1121589 |
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| Submission date |
Jun 11, 2024 |
| Last update date |
Aug 08, 2024 |
| Contact name |
Shu Xu |
| E-mail(s) |
shuxu@cpu.edu.cn
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| Organization name |
China Pharmaceutical University
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| Street address |
China Pharmaceutical University
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| City |
Nanjing |
| State/province |
Jiangsu Province |
| ZIP/Postal code |
210009 |
| Country |
China |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (6)
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| Supplementary file |
Size |
Download |
File type/resource |
| GSE269592_RAW.tar |
940.0 Kb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
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