CN105400898A - Detection technology for guiding non-small cell lung cancer appropriate radiotherapy dose through plasma miRNA level - Google Patents
Detection technology for guiding non-small cell lung cancer appropriate radiotherapy dose through plasma miRNA level Download PDFInfo
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Abstract
The invention discloses a detection technology for guiding the non-small cell lung cancer appropriate radiotherapy dose through the plasma miRNA level. The technology comprises the steps that 1, peripheral blood of a lung cancer patient is drawn before radiotherapy, and plasma is extracted; 2, plasma total RNA is separated and purified; 3, cel-miR-39-3p(cel-39) is used as a reference and is subjected to reverse transcription along with a miR-18a-5p reverse transcription primer; 4, a miR-18a-5pPCR primer is added for carrying out 10-cycle pre-amplification; 5, a cel-39 primer and a miR-18a-5pPCR primer are added, and a qPCR method is adopted for detecting the plasma miR-18a-5p level of the lung cancer patient; 6, the miRNA interpretation standard is that miR-18a-5p/cel-39 is larger than 2.28. The design is reasonable, miRNAs in plasma can reflect the biological characteristics of cancer cells, the miRNA expression level in plasma is detected, and therefore lung cancer radiosensitivity is predicted. The detection technology has the advantages of being rapid, concise and accurate.
Description
Technical field
The invention belongs to medicine bioengineering invention technical field, specifically, relate to a kind of blood plasma miRNA level and instruct nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry.
Background technology
Lung cancer is one of malignant tumour that sickness rate and mortality ratio are the highest, and its main histological type is nonsmall-cell lung cancer (non-smallcelllungcancer, NSCLC).Radiotherapy is the critical treatment means of lung cancer, and according to statistics, the NSCLC patient of 60%-70% need accept radiotherapy in the different steps of disease.
Even but radical radiation therapy thoroughly can not eliminate tumour, conventional fractionated radiation therapy (conventionalfractionatedradiotherapy, CFRT) 2 years recurrence rates are up to 60%-70%, and improving radiotherapy in lung cancer curative effect is further need the urgent problem solved at present.Radioresistance has a strong impact on radiotherapy effect, assesses before radiotherapy to patient irradiation's sensitivity, contributes to working out best radiotherapy planning, improves radiotherapy effect, but responsive, the effective outcome prediction index of current clinical shortage.
Microrna (microRNA, miRNA) is the endogenous non-coding tiny RNA that a class is guarded, in the expression of post-transcriptional level regulation and control target gene.Important regulating and controlling effect is all played in the generation, evolution of kinds of tumors, current research finds, it is also closely related that itself and chemicotherapy are resisted, tumor stem cell characteristic maintains, epithelial-mesenchymal transforms biological properties such as (Epithelial-mesenchymaltransition, EMT).MiRNA expresses has tissue specificity, and in blood plasma, Absorbable organic halogens is expressed, and can carry out quantitative detecting analysis, and therefore blood plasma miRNA is expected to the Novel index becoming radiotherapy in lung cancer outcome prediction.
Comparatively speaking, as technician, developing a kind of blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, reasonable in design, blood plasma miRNA can reflect the biological characteristics of tumour cell, detects blood plasma miRNA expression level, thus prediction radiotherapy in lung cancer susceptibility, have fast, succinctly, advantage accurately, to the clinical application of radiotherapy in lung cancer, there is important directive significance.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, reasonable in design, blood plasma miRNA can reflect the biological characteristics of tumour cell, detect blood plasma miRNA expression level, thus prediction radiotherapy in lung cancer susceptibility, have fast, succinctly, advantage accurately.
For solving the problem, the technical solution adopted in the present invention is:
Blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that:
(1), extract peripheral blood before Patients with Lung Cancer in Radiotherapy, extract blood plasma;
(2), separation and purification blood plasma total serum IgE;
(3), using cel-miR-39-3p (cel-39) as External reference, reverse transcription is carried out together with miR-18a-5p reverse transcription primer;
(4) the pre-amplification that miR-18a-5pPCR primer carries out 10 circulations, is added;
(5), cel-39 primer and miR-18a-5pPCR primer is added, qPCR method detection of lung cancer patients blood plasma miR-18a-5p level;
(6), miRNA interpretation standard: miR-18a-5p/cel-39>2.28.
As a kind of technical scheme of optimization, the method for step (2) separation and purification blood plasma total serum IgE is as follows:
1) 200 μ l blood plasma are got in 1.5mlEP pipe;
2) in EP pipe, add 1000 μ lQIAzolLysisReagent lysates, rotate mixing;
3) ambient temperatare puts 5min;
4) add 200 μ l trichloromethanes, rotate mixing 15s;
5) ambient temperatare puts 2-3min;
6) 4 DEG C, the centrifugal 15min of 12000g;
7) supernatant liquor (600 μ l) is transferred in new 1.5mlEP pipe, add 900 μ l dehydrated alcohols;
8) the mixing liquid 750 μ l of step 8 is added 2mlRNeasyMinEluteSpinColumns, the centrifugal 15s of room temperature 10000rpm, abandons lower floor's liquid;
9) repeating step 8);
10) in 2mlRNeasyMinEluteSpinColumns, add 700 μ lRWTbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
11) in 2mlRNeasyMinEluteSpinColumns, add 500 μ lRPEbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
12) in 2mlRNeasyMinEluteSpinColumns, add 500 μ l80% dehydrated alcohols, the centrifugal 2min of 10000rpm, abandons lower floor's liquid;
13) wash-out film is implanted new 2ml collection tube, the centrifugal 5min of top speed;
14) wash-out film is implanted in new 1.5ml collection tube, add 20 μ l without enzyme water, the centrifugal 1min of top speed;
15), after centrifugal, in collection tube, total serum IgE is;
16) ultramicrospectrophotometer is used to measure the total rna concentration extracted.
As a kind of technical scheme of optimization, the method for step (3) reverse transcription is:
1) reverse transcription liquid (20 μ l) is configured:
5×PrimeScriptBuffer，4μl；
PrimeScriptRTEnzymeMix，1μl；
MiR-18a-5p reverse transcription primer (2 μMs), 0.5 μ l;
Cel-39 reverse transcription primer (2 μMs), 0.5 μ l;
cel-39standard(0.1μM)，1μl；
Total serum IgE (100ng), 1 μ l
Without enzyme water, supply 20 μ l.
2) reaction conditions:
Step1：42℃，15min；
Step2：85℃，5s。
The pre-amplification method of step (4) is:
1) liquid (12.5 μ l) that increases in advance is prepared
cDNA，1μl；
MiR-18a-5p upstream primer (10 μMs), 0.5 μ l;
MiR-18a-5p downstream primer (10 μMs), 0.5 μ l;
2×TaqPCRMasterMix，6.25μl；
Without enzyme water, 4.25 μ l;
2) reaction conditions (regular-PCR instrument)
Reps：94℃，3min；
Reps：94℃，30s；60℃，30s；72℃，1min；10cycles；
Reps：72℃，5min。
As a kind of technical scheme of optimization, step (5) qPCR method is:
1) PCR reaction solution (20 μ l system) is prepared
SYBRPremixExTaqⅡ，10μl；
Upstream primer (10uM), 0.5 μ l;
Downstream primer (10uM), 0.5 μ l;
ROXⅡ，0.4μl；
Pre-amplification liquid, 2 μ l;
Without enzyme water, 6.6 μ l;
2) reaction conditions
Reps：95℃，30s；
Reps：95℃，5s；60℃，34s；40cycles。
As a kind of technical scheme of optimization, interpretation standard: miR-18a-5p/cel-39>2.28 in step (6).
Owing to have employed technique scheme, the Sensitivity and Specificity difference 87% and 95% of miR-18a-5p in this detection technique of the present invention.The patient of miR-18a-5p/cel-39>2.28, gives lower Radiotherapy dosimetry (56-60Gy), the patient of miR-18a-5p/cel-39≤2.28, gives higher Radiotherapy dosimetry (66-70Gy).
The present invention is reasonable in design, and blood plasma miR-18a-5p can reflect the biological characteristics of tumour cell, detects blood plasma miR-18a-5p level, prediction Patients with Lung Cancer in Radiotherapy susceptibility, have fast, succinctly, advantage accurately.
Embodiment
Embodiment:
Blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, and total method is as follows:
(1), extract peripheral blood before Patients with Lung Cancer in Radiotherapy, extract blood plasma;
(2), separation and purification blood plasma total serum IgE;
(3), using cel-miR-39 (cel-39) as External reference, reverse transcription is carried out together with miR-18a-5p reverse transcription primer;
(4) the pre-amplification that miR-18a-5pPCR primer carries out 10 circulations, is added;
(5), cel-39 primer and miR-18a-5pPCR primer is added, qPCR method detection of lung cancer patients blood plasma miR-18a-5p level;
(6), miRNA examination criteria: miR-18a-5p/cel-39>2.28.
Specifically, the method for step (2) separation and purification blood plasma total serum IgE is:
1) 200 μ l blood plasma are got in 1.5mlEP pipe;
2) in EP pipe, add 1000 μ lQIAzolLysisReagent lysates, rotate mixing;
3) ambient temperatare puts 5min;
4) add 200 μ l trichloromethanes, rotate mixing 15s;
5) ambient temperatare puts 2-3min;
6) 4 DEG C, the centrifugal 15min of 12000g;
7) supernatant liquor (600 μ l) is transferred in new 1.5mlEP pipe, add 900 μ l dehydrated alcohols;
8) the mixing liquid 750 μ l of step 8 is added 2mlRNeasyMinEluteSpinColumns, the centrifugal 15s of room temperature 10000rpm, abandons lower floor's liquid;
9) repeating step 8);
10) in 2mlRNeasyMinEluteSpinColumns, add 700 μ lRWTbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
11) in 2mlRNeasyMinEluteSpinColumns, add 500 μ lRPEbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
12) in 2mlRNeasyMinEluteSpinColumns, add 500 μ l80% dehydrated alcohols, the centrifugal 2min of 10000rpm, abandons lower floor's liquid;
13) wash-out film is implanted new 2ml collection tube, uncap, the centrifugal 5min of top speed;
14) wash-out film is implanted in new 1.5ml collection tube, add 20 μ l without enzyme water, the centrifugal 1min of top speed;
15), after centrifugal, in collection tube, total serum IgE is;
16) ultramicrospectrophotometer is used to measure the total rna concentration extracted.
The method of step (3) reverse transcription is:
1) reverse transcription liquid (20 μ l) is configured:
5×PrimeScriptBuffer，4μl；
PrimeScriptRTEnzymeMix，1μl；
MiR-18a-5p reverse transcription primer (2 μMs), 0.5 μ l;
Cel-39 reverse transcription primer (2 μMs), 0.5 μ l;
cel-39standard(0.1μM)，1μl；
Total serum IgE (100ng), 1 μ l
Without enzyme water, supply 20 μ l.
2) reaction conditions:
Step1：42℃，15min；
Step2：85℃，5s。
The method that step (4) increases in advance is:
1) liquid (12.5 μ l) that increases in advance is prepared
cDNA，1μl；
MiR-18a-5p upstream primer (10 μMs), 0.5 μ l;
MiR-18a-5p downstream primer (10 μMs), 0.5 μ l;
2×TaqPCRMasterMix，6.25μl；
Without enzyme water, 4.25 μ l.
2) reaction conditions (regular-PCR instrument)
Reps：94℃，3min；
Reps：94℃，30s；60℃，30s；72℃，1min；10cycles；
Reps：72℃，5min。
The method of step (5) qPCR is:
1) PCR reaction solution (20 μ l system) is prepared
SYBRPremixExTaqⅡ，10μl；
Upstream primer (10uM), 0.5 μ l;
Downstream primer (10uM), 0.5 μ l;
ROXⅡ，0.4μl；
Pre-amplification liquid, 2 μ l;
Without enzyme water, 6.6 μ l.
2) reaction conditions
Reps：95℃，30s；
Reps：95℃，5s；60℃，34s；40cycles。
MiRNA interpretation standard in step (6):
miR-18a-5p/cel-39>2.28。
The Sensitivity and Specificity difference 87% and 95% of miR-18a-5p in this detection technique of the present invention.The patient of miR-18a-5p/cel-39>2.28, can give lower Radiotherapy dosimetry (56-60Gy), the patient of miR-18a-5p/cel-39≤2.28, need give higher Radiotherapy dosimetry (66-70Gy).
The present invention is reasonable in design, and blood plasma miR-18a-5p can reflect the biological characteristics of tumour cell, detects blood plasma miR-18a level, prediction Patients with Lung Cancer in Radiotherapy susceptibility, have fast, succinctly, advantage accurately.
The present invention is not limited to above-mentioned preferred forms, and anyone should learn the structural changes made under enlightenment of the present invention, and every have identical or akin technical scheme with the present invention, all belongs to protection scope of the present invention.
Claims (6)
1. blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: step is as follows:
(1), extract peripheral blood before Patients with Lung Cancer in Radiotherapy, extract blood plasma;
(2), separation and purification blood plasma total serum IgE;
(3), using cel-miR-39-3p (cel-39) as External reference, reverse transcription is carried out together with miR-18a-5p reverse transcription primer;
(4) the pre-amplification that miR-18a-5pPCR primer carries out 10 circulations, is added;
(5), cel-39 primer and miR-18a-5pPCR primer is added, qPCR method detection of lung cancer patients blood plasma miR-18a-5p level;
(6), miRNA interpretation standard: miR-18a-5p/cel-39>2.28.
2. according to claim 1, blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: the method for step (2) separation and purification blood plasma total serum IgE is as follows:
1) 200 μ l blood plasma are got in 1.5mlEP pipe;
2) in EP pipe, add 1000 μ lQIAzolLysisReagent lysates, rotate mixing;
3) ambient temperatare puts 5min;
4) add 200 μ l trichloromethanes, rotate mixing 15s;
5) ambient temperatare puts 2-3min;
6) 4 DEG C, the centrifugal 15min of 12000g;
7) supernatant liquor (600 μ l) is transferred in new 1.5mlEP pipe, add 900 μ l dehydrated alcohols;
8) the mixing liquid 750 μ l of step 8 is added 2mlRNeasyMinEluteSpinColumns, the centrifugal 15s of room temperature 10000rpm, abandons lower floor's liquid;
9) repeating step 8);
10) in 2mlRNeasyMinEluteSpinColumns, add 700 μ lRWTbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
11) in 2mlRNeasyMinEluteSpinColumns, add 500 μ lRPEbuffer, the centrifugal 15s of 10000rpm, abandons lower floor's liquid;
12) in 2mlRNeasyMinEluteSpinColumns, add 500 μ l80% dehydrated alcohols, the centrifugal 2min of 10000rpm, abandons lower floor's liquid;
13) wash-out film is implanted new 2ml collection tube, the centrifugal 5min of top speed;
14) wash-out film is implanted in new 1.5ml collection tube, add 20 μ l without enzyme water, the centrifugal 1min of top speed;
15), after centrifugal, in collection tube, total serum IgE is;
16) ultramicrospectrophotometer is used to measure the total rna concentration extracted.
3. according to claim 1, blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: the method for step (3) reverse transcription is:
1) reverse transcription liquid (20 μ l) is configured:
5×PrimeScriptBuffer，4μl；
PrimeScriptRTEnzymeMix，1μl；
MiR-18a-5p reverse transcription primer (2 μMs), 0.5 μ l;
Cel-39 reverse transcription primer (2 μMs), 0.5 μ l;
cel-39standard(0.1μM)，1μl；
Total serum IgE (100ng), 1 μ l
Without enzyme water, supply 20 μ l.
2) reaction conditions:
Step1：42℃，15min；
Step2：85℃，5s。
4. according to claim 1, blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: the pre-amplification method of step (4) is:
1) liquid (12.5 μ l) that increases in advance is prepared
cDNA，1μl；
MiR-18a-5p upstream primer (10 μMs), 0.5 μ l;
MiR-18a-5p downstream primer (10 μMs), 0.5 μ l;
2×TaqPCRMasterMix，6.25μl；
Without enzyme water, 4.25 μ l;
2) reaction conditions (regular-PCR instrument)
Reps：94℃，3min；
Reps：94℃，30s；60℃，30s；72℃，1min；10cycles；
Reps：72℃，5min。
5. according to claim 1, blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: step (5) qPCR method is:
1) PCR reaction solution (20 μ l system) is prepared
SYBRPremixExTaqⅡ，10μl；
Upstream primer (10uM), 0.5 μ l;
Downstream primer (10uM), 0.5 μ l;
ROXⅡ，0.4μl；
Pre-amplification liquid, 2 μ l;
Without enzyme water, 6.6 μ l;
2) reaction conditions
Reps：95℃，30s；
Reps：95℃，5s；60℃，34s；40cycles。
6. according to claim 1, blood plasma miRNA level instructs nonsmall-cell lung cancer to be suitable for the detection technique of Radiotherapy dosimetry, is characterised in that: interpretation standard: miR-18a-5p/cel-39>2.28 in step (6).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110189798A (en) * | 2019-06-26 | 2019-08-30 | 广州市雄基生物信息技术有限公司 | A kind of clustering method and application based on peripheral blood plasma DNA nucleosome footprint difference |
| CN112301130A (en) * | 2020-11-12 | 2021-02-02 | 苏州京脉生物科技有限公司 | Marker, kit and method for early detection of lung cancer |
-
2015
- 2015-12-24 CN CN201510989691.7A patent/CN105400898A/en active Pending
Non-Patent Citations (4)
| Title |
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| 吴磊等: "miR-18a对A549细胞的放射增敏作用及其机制", 《第三军医大学学报》 * |
| 徐燕梅: "血浆miRNAs预测非小细胞肺癌患者放疗敏感性的研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 朱龙萍等: "食管鳞癌肿瘤微环境中miRNA与TGF-β1的相关性分析", 《细胞与分子免疫学杂志》 * |
| 陈昌国等: "三种方法提取血清miRNA效果的比较", 《检验医学与临床》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110189798A (en) * | 2019-06-26 | 2019-08-30 | 广州市雄基生物信息技术有限公司 | A kind of clustering method and application based on peripheral blood plasma DNA nucleosome footprint difference |
| CN112301130A (en) * | 2020-11-12 | 2021-02-02 | 苏州京脉生物科技有限公司 | Marker, kit and method for early detection of lung cancer |
| CN112301130B (en) * | 2020-11-12 | 2021-11-30 | 苏州京脉生物科技有限公司 | Marker, kit and method for early detection of lung cancer |
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