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Reagent kit for predicting semen collection outcome of patient suffering from non-obstructive azoospermia

An azoospermia and kit technology, applied in the field of biomedicine, can solve the problems of poor stability, poor sensitivity, and difficult clinical application of free RNA detection, and achieve fully scientific and practical effects.

Active Publication Date: 2020-11-17
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large-scale clinical study involving 1371 patients with NOA showed that the area under the curve (AUC) of the clinical prediction model based on FSH, LH and testosterone was only 0.67; The detection sensitivity is poor; the detection stability of free RNA in semen is poor; testicular tissue samples can be obtained by testicular biopsy, but as an invasive examination method, it is difficult to achieve a wide range of clinical applications

Method used

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  • Reagent kit for predicting semen collection outcome of patient suffering from non-obstructive azoospermia
  • Reagent kit for predicting semen collection outcome of patient suffering from non-obstructive azoospermia
  • Reagent kit for predicting semen collection outcome of patient suffering from non-obstructive azoospermia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Purification and long-chain RNA sequencing of seminal exosomes from normal volunteers and NOA patients

[0026] Take semen (>0.5 mL) from a single sample, centrifuge at 4°C, 12,000×g for 30 minutes to remove cell debris, then take the supernatant, filter it through a 0.22 μm filter, and collect exocytosis by ultracentrifugation at 4°C, 100,000×g for 70 minutes Body precipitation. After the pellet was resuspended in PBS, it was again ultracentrifuged at 100,000×g at 4°C for 70 min to obtain purified exosome pellet.

[0027] The purified exosomes were precipitated, added to the RNA lysate, and the total exosome RNA was obtained by phenol-chloroform extraction and library construction. Followed by long-chain RNA (lncRNA) sequencing.

Embodiment 2

[0028] Example 2 Target gene screening

[0029] Analyze the sequencing data in Example 1, screen the testis-specific highly expressed genes according to the NCBI database information, and select the top 20 genes showing a downward trend in the sequencing results and the largest log2(FC) multiple of difference.

Embodiment 3

[0030] Example 3 Determination of lncRNA expression in seminal plasma exosomes of NOA patients

[0031] The exosomal RNA in the seminal plasma of NOA patients was extracted and detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The specific steps are as follows:

[0032] (1) Precipitate the purified exosomes obtained in Example 1, add RNA lysate, obtain exosome total RNA by phenol-chloroform extraction, and use Nanodrop2000 to detect RNA concentration and purity. The reverse transcription reaction was performed with 500ng RNA / 10 μL system, and the reverse transcription reaction solution was obtained and its concentration was detected. Dilute the reverse transcription reaction solution to 10 ng / μL with DEPC water, aliquot 20 μL / tube and store at -80°C. For the reverse transcription reaction solution used for qRT-PCR detection, the concentration of the reverse transcription reaction solution should be adjusted to 2.5ng / μL.

[0033](2) Using β-A...

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Abstract

The invention relates to the field of biological medicine, in particular to a reagent kit for predicting semen collection outcome of a patient suffering from non-obstructive azoospermia. The reagent kit comprises a reagent for detecting the expression level of lncRNA in a sample, and the lncRNA comprises at least one of SPATA42, CCDC37-DT, GABRG3-AS1, LOC440934, LOC100505685, LOC101929088 (XR_927561.2), LOC101929088 (XR_001745218.1), LINC00343 and LINC00301. According to the reagent kit, the sperm extraction outcome of the patient suffering from NOA can be noninvasively and accurately predicted before an operation, and the reagent kit has sufficient scientificity and practicability.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a kit for predicting the outcome of sperm retrieval in patients with non-obstructive azoospermia. Background technique [0002] Non-obstructive azoospermia (NOA) is the most serious male infertility, and its treatment has always been a hot and difficult point in the field of reproductive research at home and abroad. Although sperm cannot be found in the semen, some patients with NOA can meet their fertility needs through testicular sperm aspiration. However, the existing technology is difficult to predict the outcome of sperm retrieval before surgery, resulting in patients not receiving optimal clinical treatment (selecting patients who are really suitable for surgery). [0003] At present, the markers used to predict the outcome of sperm retrieval in patients with NOA before surgery mainly come from blood, semen, and testicular tissue. Among the above samples, the detection of hormo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883G16B25/10
CPCC12Q1/6883G16B25/10C12Q2600/158C12Q2600/178
Inventor 刘贵华谢云邹学农邓春华
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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