Application of reagent used for detecting expression level of long-chain non-coding RNA LNC_004208 in preparing brain glioma diagnostic reagent
A technology of diagnostic reagents and detection reagents, which is applied in the field of tumor molecular biology, can solve problems such as the specific mechanism has not been clarified, and achieve the effect of important promotion and application prospects and far-reaching clinical significance
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Embodiment 1
[0029] Example 1 Transcriptome sequencing technology and lncRNA sequencing technology analysis of TMZ sensitive / resistant cell expression profile
[0030] Transcriptome sequencing technology and lncRNA sequencing technology (Illumina HiSeq TM 2000), selected brain glioma T98G / U-118MG cell line and TMZ-resistant T98G-R / U-118MG-R cell line for expression profile analysis. Before screening, we first used Cuffmerge software to merge the transcripts spliced from each sample, and removed transcripts with uncertain strand orientations to obtain the complete transcriptome information for this sequencing. The screening process is divided into the following 5 steps ( figure 1 A):
[0031] step1: Screening of the number of transcript exons: filter a large number of low-expression, low-confidence single-exon transcripts in the transcriptome splicing results, and select transcripts with exon numbers >= 2 (if there is no special requirement, the plant will Transcripts with exon number...
Embodiment 2
[0042] Example 2 Screening and verification of differentially expressed lncRNAs in glioma TMZ-resistant cells
[0043] (1) Extraction of total cellular RNA (TRIzol method)
[0044] Cells were seeded in 6-well plates at 37°C, 5% CO 2 Incubate in the incubator. After the cells were completely attached to the wall, 1000 μl TRIzol was added to each well. After standing still until completely dissolved, transfer to a 1.5ml EP tube. Add 0.2ml of chloroform, shake vigorously for 15 seconds, and place at room temperature for 3 minutes. Centrifuge at 4°C and 12000rpm for 15min, and transfer the supernatant to a new EP tube. Add 0.5ml of isopropanol and let stand at room temperature for 10min. After centrifugation at 4°C and 12,000 rpm for 10 minutes, a gelatinous precipitate appeared at the bottom of the EP tube. Discard the supernatant, wash the RNA pellet with 1ml of 75℅ ethanol, and centrifuge at 4°C, 7500rpm for 5min, discard the supernatant again. After air-drying for 5 min...
Embodiment 3
[0060] Example 3 Correlation between glioma grade and lncRNA expression
[0061] The grading of gliomas is based on astrocytomas and further grading according to their malignancy. The general WHO grading is divided into 4 grades based on atypicality, mitotic index, endothelial cell proliferation and necrosis: grade 1, generally benign and Pilocytic astrocytoma dominates, accounting for about 5% of gliomas and can be cured; grade 2, general astrocytoma or astrocytoma-oligodendroglioma, accounting for 30-40% of gliomas about. Grade 3, interstitial astrocytoma, accounts for about 15-25% of gliomas, and generally evolves from grade 2. Grade 4 is glioblastoma, accounting for about 1 / 3 of gliomas. Gliomas can be further classified according to the pathological malignancy of tumor cells. Low-grade glioma (WHO grade 1-2) is a well-differentiated glioma; although this type of tumor is not biologically benign, the prognosis of patients is relatively good. High-grade gliomas (WHO gra...
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