Clinic-prognosis-related cytokines in blood of EBOV infected patient and application
A technology for infection, prognosis, applied in the field of biomedicine
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Embodiment 1
[0050] Example 1. Molecular markers related to viral load in the blood of EBOV-infected patients
[0051] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use magnetic beads with Oligo(dT) to absorb and purify the mRNA in the total RNA, fragment the mRNA under heating conditions, and use this as a template for inversion Recorded into double-stranded cDNA. The double-stranded cDNA is repaired and filled to phosphorylate the 5' end and add "A" to the 3' end. The double-stranded cDNA adapter with 3'dTMP end was connected to the sequencing adapter, amplified by PCR, enriched and purified with AMPure XP magnetic beads, and the library was identified by PCR reaction. The constructed library was subjected to paired-end sequencing using the Illumina sequencing platform. Use the Perl script to filter adapter sequences, low-quality sequences at both ends (sequences with Q<=20 bases accounting for more than 50% of the entire reads), and l...
Embodiment 2
[0057] Embodiment 2, small RNAs (sRNAs) associated with viral load in the blood of EBOV-infected patients
[0058] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use 15% denatured polyacrylamide gel electrophoresis (PAGE) to separate small fragments (15-30nt) RNA from total RNA, and isolate small fragments after purification The fragmented RNA was ligated with adapters at the 3' and 5' ends, and then the RNA with the adapters was reverse-transcribed using SuperScriptII reverse transcriptase (product of Invitrogen) to synthesize cDNA. Then, PCR amplification was carried out, and the amplification program was: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 60°C for 30s, extension at 72°C for 15s, 14 cycles, and extension at 72°C for 8min, thereby constructing the library. Finally, the extended products were subjected to high-throughput sequencing using the Illumina sequencing platform. Use the Perl ...
Embodiment 3
[0064] Example 3. Molecular markers related to clinical prognosis in the blood of EBOV-infected patients
[0065] Extract total RNA from the whole blood of EBOV-infected patients and non-EBOV-infected patients, use magnetic beads with Oligo(dT) to absorb and purify the mRNA in the total RNA, fragment the mRNA under heating conditions, and use this as a template for inversion Recorded into double-stranded cDNA. The double-stranded cDNA is repaired and filled to phosphorylate the 5' end and add "A" to the 3' end. The double-stranded cDNA adapter with 3'dTMP end was connected to the sequencing adapter, amplified by PCR, enriched and purified with AMPure XP magnetic beads, and the library was identified by PCR reaction. The constructed library was subjected to paired-end sequencing using the Illumina sequencing platform. Use the Perl script to filter adapter sequences, low-quality sequences at both ends (sequences with Q<=20 bases accounting for more than 50% of the entire reads...
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