CN102071253A - Method for detecting microRNA expression level in excrement and selection method of internal reference gene - Google Patents
Method for detecting microRNA expression level in excrement and selection method of internal reference gene Download PDFInfo
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Abstract
The invention discloses a method for detecting the microRNA expression level in excrement. The method mainly comprises the following steps: extracting with an excrement extraction kit from a collected excrement specimen, identifying the concentration of RNA with a NanoDrop; performing reverse transcription to generate cDNA, detecting the expression of the target microRNAs through Real-time RT-PCR; and adopting Real Time PCR to obtain the CT value of each microRNA, adopting the -Delta Delta Ct method to calculate the expression abundance of each microRNA, and selecting an internal reference gene. The invention also provides a selection method of the internal reference gene used for detecting pancreatic cancer. The detection method provided by the invention has reliable result and good stability.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of the detect method of microRNA expression level in the ight soil and the system of selection of internal control gene.
Background technology
MicroRNA (miRNA) molecule is the research focus in molecular biology and genetics field in recent years.MiRNA is the newfound non-coding small RNA molecular of a class, usually at the post-transcriptional level regulate gene expression.The miRNA gene is transcribed earlier and is produced the elementary product (pri-miRNA) of hundreds of to several thousand Nucleotide, in nuclear, cut into 70 hair clip shape precursors (pre-miRNA) about Nucleotide by the Drosha enzyme of RNaseIII restriction endonuclease family, pre-miRNA transports out nuclear under the effect of transhipment egg Exortin=5, then through ripe miRNA (the Gregory RI of Dicer enzyme cutting into about 22nt, Shlekhattar R.MicroRNA biogenesisand cancer.Cancer Res, 2005,65 (9): 3509-3512.).Sophisticated miRNA induces reticent mixture (RNA-induced silencing complex by forming RNA, RISC), mate fully or not exclusively with said target mrna and to combine, induce said target mrna degraded or check it and transcribe the back translation, thereby regulate cell proliferation, differentiation and apoptosis, participate in processes such as ontogeny, organism metabolism and tumour generation, development.
In the past discover protein coding gene can cause developing of tumour unusually; the reported first miRNA of Croce study group is unusual relevant with tumour from November, 2002, and more and more evidences demonstration miRNA plays an important role in the developing of tumour.There are some experiments and clinical analysis to point out that miRNAs plays a role as one of oncogene or cancer suppressor gene new classification.Those express the oncogene thought that raises in tumour, these oncogene miRNAs promote the development of tumour usually by the gene of negative regulation tumor suppressor gene or control cytodifferentiation or apoptosis.Found many miRNAs obvious expression excessively in different tumours.
The method that detects the microRNA expression level at present mainly comprises microarray, RT-PCR/Northern blot, Ribonulease protection assay (RPA), Bead-based flow cytometric assay (detection of magnetic bead streaming), in situ hybridization etc.Because the miRNA sequence is very short, has increased detection difficulty.The Step-loop real-time quantitative RT-PCR that this institute uses is the experimental technique that the detection miRNA of a high specific degree, susceptibility expresses, and comprises that design has the reverse transcription primer of stem ring (stem-loop) structure and carries out 2 committed steps of PCR in real time with the fluorescently-labeled special molecular probe of miRNA.Shi (Shi R, ChiangVL.Facile means for quantifying microRNA expression by real time PCR[J] .Biotechniques, 2005,39 (4): 519-525 etc. are used for real-time quantitative RT-PCR to RNA tailing and primer extension, have successfully detected miRNA expression in the plant.Zhang Qi (Zhang Qi, He Xiangjun, Pan Xiu English .RNA tailing and primer extension RT-PCR method real-time quantitative detect microRNA[J]. Peking University's journal, 2007,39 (1): 87-91) grade uses the same method 15 kinds of miRNA is detected in the rat heart, liver and corticocerebral expression, has verified the reliability of this technology.
Gene test has caused that owing to its wide application prospect people pay attention in the ight soil at present,, but its used detection method difference, comparability is relatively poor each other.Therefore need seek new molecular marker on the one hand, improve detection method and technology on the other hand, improve detection sensitivity, can be non-invasive carcinoma of the pancreas examination and diagnosing provides new selection.
Studies show that based on above-mentioned, the research of relevant microRNA is confined to hemopathy mostly, lung cancer, hepatocellular carcinoma, mammary cancer, colorectal cancer, research to carcinoma of the pancreas is very few, in the research of carcinoma of the pancreas, also only only limit to tissue, blood equal samples, and the microRNA that be not reported in the ight soil detects.Universally applicable house-keeping gene is non-existent in fact, find out suitable house-keeping gene as confidential reference items accurately quantitatively most important for gene expression analysis, at present mostly by using the geNorm program, select from candidate's house-keeping gene of a series of difference in functionalitys that the house-keeping gene of suitable number is used for the stdn of target gene, thereby can reach accurate quantitative experiment purpose.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method that detects microRNA expression level in the ight soil, and this method is formulated a kind of method of calculation and presented the differential expression of different microRNA in ight soil by selecting the internal control gene of suitable confidential reference items.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
A kind of method that detects microRNA expression level in the ight soil mainly may further comprise the steps:
(1) stool sample of collecting is extracted with ight soil extraction agent box, extract RNA is identified in the back with NanoDrop concentration;
(2) will identify that good RNA carries out reverse transcription and generates cDNA, by the expression of Real-time RT-PCR testing goal microRNA;
(3) obtain the CT value of each microRNA by Real Time PCR, calculate the expression abundance of each microRNA by-Δ Δ Ct method again, calculation formula is-Δ Δ Ct=-{ (Sample Cttarget-Sample CtmiR-16)-(ControlCttarget-Control CtmiR-16) };
(4) choose internal control gene, pick out the purpose differential gene.
Another technical issues that need to address of the present invention provide a kind of system of selection that is used to detect the internal control gene of carcinoma of the pancreas.
The technical scheme that solves the problems of the technologies described above is as follows:
A kind of system of selection that is used to detect the internal control gene of carcinoma of the pancreas mainly may further comprise the steps:
(1) stool sample with the Pancreas cancer patients of collecting extracts with ight soil extraction agent box, extracts the concentration of back with NanoDrop evaluation RNA;
(2) will identify that good RNA carries out reverse transcription and generates cDNA, by the expression of Real-time RT-PCR testing goal microRNA;
(3) obtain the CT value of each microRNA, calculate the expression abundance of each microRNA by-Δ Δ Ct method again, calculation formula is-Δ Δ Ct=-(Ct
Target microRNAs-Ct
MiR-16);
(4) choose suitable internal control gene.
The present invention detects microRNA expression level in the ight soil by the real-time quantitative method, finds that extracting RNA (comprising microRNA) method is reliable and stable in the ight soil, can be used for real-time quantitative and detect.The detected result of real-time quantitative is chosen the internal control gene of stably express by data input Genorm software, and final passing through-Δ Δ Ct method picked out the purpose differential gene.
The method of microRNA expression level can overcome the shortcoming of existing method fluctuation of service in the detection ight soil of the present invention, thereby can be used for simply, stably detect in the ight soil expression and the difference of microRNAs, the gained result is reliable and stable, and is repeatable high.
A kind of system of selection that is used to detect the internal control gene of carcinoma of the pancreas of the present invention, the total microRNAs of extracting from ight soil, detect through real time quantitative PCR method then, screening obtains confidential reference items miRNA16, the detection of the relevant ight soil microRNA relative content of its suitable pancreatic disease patient.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Figure 1A is the repeatable comparative result synoptic diagram of embodiment of the invention 1total RNA extracting,
Figure 1B is that embodiment 1 part sample microRNA detects repeatable result schematic diagram;
Fig. 2 is the selection of carcinoma of the pancreas internal control gene of the present invention;
Fig. 3 is the microRNAs figure of differential expression in the embodiment of the invention 1.
Embodiment
In the following example, the experimental technique of unreceipted actual conditions, usually condition routinely, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, chief editors such as J.G. Sai Deman, Ma Xuejun, the Su Yuelong translates. Beijing: Science Press, 2004) described in method carry out.
Now the detection with Pancreas cancer patients ight soil microRNAs is an example, and detailed description is done in invention.This embodiment only is used to the present invention is described and is not used in and limits the scope of the invention.
Choose the 29 routine carcinoma of the pancreas that Changhai hospital hospitalized in 2008 to 2010,22 routine chronic pancreatitis patients, and collect this laboratory normal healthy controls 13 examples.
1, specimen collection places ice chest immediately behind frozen pipe, and the Ultralow Temperature Freezer that was transferred to-80 ℃ in 6 hours is preserved.Need take by weighing from frozen pipe 200mg ight soil during the RNA extracting of ight soil, the extracting of total RNA (comprising microRNA) is extracted total microRNAs, concrete steps by E.Z.N.A ight soil extraction agent box (U.S., OMEGA company)
As follows:
Take by weighing the 200mg fecal sample to the 2ml centrifuge tube that the 200mg glass bead is housed
↓
Add 500ul Buffer RPL and vibration mixing
↓
Add saturated phenol, 65 ℃ of water-bath 10min behind the vibration mixing
↓
Add the 500ul chloroform, vibrate 30 seconds to thorough mixing
↓
Placed 5 minutes on ice
↓
Centrifugal 5 minutes of 12000rpm
↓
The careful new 2ml centrifuge tube of 500ul supernatant to of drawing is not run into the throw out in the centrifuge tube
↓
Add 500ul RB and 500ul raw spirit, the vibration mixing
↓
The aforesaid liquid that adds 750ul is to HiBind RNA pillar, and under the 12000rpm room temperature centrifugal 30 seconds,
Discard collection tube
↓
The repetition previous step is rapid, and another 750ul liquid is added HiBind RNA pillar
↓
Draw 500ul RWC Buffer to centrifugal post, under the 12000rpm room temperature centrifugal 30 seconds, discard collection tube
↓
Draw 500ul RWB Buffer to centrifugal post, under the 10000rpm room temperature centrifugal 30 seconds, discard collection tube
↓
Draw 500ul RWB Buffer to centrifugal post, under the 10000rpm room temperature centrifugal 1 minute, discard collection tube
↓
HiBind RNA pillar is inserted in the new 2ml centrifuge tube, under the 12000rpm room temperature centrifugal 2 minutes complete until film
Dry
↓
Add 50ul DEPC water in above-mentioned pillar, room temperature was placed 2 minutes
↓
Under the 10000rpm room temperature centrifugal 1 minute
Temperature centrifugal 30 seconds down discards collection tube
↓
Draw 500ul RWB Buffer to centrifugal post, under the 10000rpm room temperature centrifugal 1 minute, discard collection tube
↓
HiBind RNA pillar is inserted in the new 2ml centrifuge tube, under the 12000rpm room temperature centrifugal 2 minutes complete until film
Dry
↓
Add 50ul DEPC water in above-mentioned pillar, room temperature was placed 2 minutes
↓
Under the 10000rpm room temperature centrifugal 1 minute.
2, the evaluation of RNA
Get the extractive RNA of 1ul and carry out 1% agarose gel electrophoresis.DNA concentration detection use NanoDrop (ND-1000, NanoDrop, Wilmington, DE, USA), record A260/A280 value and concentration value.Purity is identified-80 ℃ of preservations in back.
Ight soil RNA concentration range is from 386.3-3399.9ng/ul, detect repeatability in order to assess ight soil RNA extracting and microRNAs, choose 6 samples and carry out revision test, show as Figure 1A, B, twice extracting and detection are linear dependence (R2=0.998, p＜0.001; R2=0.988, p＜0.001).
3, the synthetic cDNA of reverse transcription
Adopt American AB I company's T aqMan MicroRNA Reverse Transcription Kit to carry out reverse transcription and generate cDNA, expression by Real-time RT-PCR testing goal microRNA, wherein, following reverse transcriptase primer and PCR primer provide by ABI company.
Reaction system is as follows:
dNTP?mix(100mM?total) 0.15ul
Multiscribe?RT?enzyme(50U/ul) 1ul
10×RT?buffer 1.5ul
RNase?inhibitor(20U/ul) 0.19ul
Nuclease?free?water 8.16ul
primer 3ul
RNA 1ul
Total 15ul
Reaction conditions is as follows:
Time(minites) Temperature(℃)
30 16
30 42
5 85
∞ 4
4, Real-time RT-PCR detects ight soil miRNAs
Reaction system is as follows:
Master?mix?II 10ul
Primer 1ul
H
2O 7ul
cDNA 2ul
Reaction conditions is as follows:
Temperature(℃) Time(minites) Reps(cycles)
Stage1 50 2 1
Stage2 95 10 1
95: 15 (15 seconds) 55 of Stage3
60 1
5, data analysis
(Applied Biosystems USA) obtains the CT value of each miRNA, calculates relative expression's abundance by following formula to utilize ABI 7500 Real Time PCR instrument SDS 2.1 software.
-ΔΔCt＝-(Ct
target?microRNAs-Ct
miR-16)
6, utilize Genorm software to select suitable internal control gene
From http://medgen.ugent.be/～jvdesomp/genorm/ website, download geNorm program, the expression amount of setting a certain house-keeping gene Ct value reckling in the different samples in Excel is 1, and the relative expression quantity of other these house-keeping genes of sample then is 2
-Δ Ct(each sample Ct value of Δ Ct=-minimum Ct value), with these data importings geNorm program, utilize this program to calculate the mean value of genetic expression stability, expression stability to house-keeping gene sorts, be worth more little, express stable more), and the pairing variance analysis (V by the house-keeping gene normalization factor
N/n+1) judge the suitableeest number of house-keeping gene.
After detecting the microRNA CT value input Genorm software of different samples, can obtain Fig. 1, the x axle is expressed as different microRNA, Y-axis is represented the stability expressed, as seen from the figure, stability increases gradually from left to right, and microRNA-196a expresses least stable, and miR-155 and miR-16 express the most stable.Finally we select miR-16 as confidential reference items.
The present invention selects 6 microRNAs (miR-155, miR-181a, miR-21, miR-181b, miR-196a and miR-210) researchs and analyses, the result shows, in carcinoma of the pancreas and normal control group, and miR-181b, miR-196a and miR-210 difference has statistical significance (p=0.03, p=0.04, p=0.01, respectively); In chronic pancreatitis and normal control group, have only the expression of miR-181b and miR-210 have than big-difference (p=0.08, p=0.05, respectively); MiR-155, miR-181a, the detection of expression of miR-21 is the difference not statistically significant in three groups of data.The diagnostic assessment of Pancreas cancer patients ight soil microRNA has only miR181b and miR-210 to have diagnostic significance by Receiver operating characteristic (ROC curve).Carcinoma of the pancreas and healthy tissues relatively in, the AUC-ROC of miR-181b and miR-210 is respectively 0.745 (95%CI:0.597-0.894) and 0.772 (95%CI:0.629-0.914), and susceptibility and specificity: miR-181b is 84.6% and 51.7% (CUTOFF value=2.52); MiR-210 is 84.6% and 65.5%. (CUTOFF value=1.54).
The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (7)
1. a method that detects microRNA expression level in the ight soil is characterized in that, mainly may further comprise the steps:
(1) stool sample of collecting is extracted with ight soil extraction agent box, extract RNA is identified in the back with NanoDrop concentration;
(2) will identify that good RNA carries out reverse transcription and generates cDNA, by the expression of Real-time RT-PCR testing goal microRNA;
(3) obtain the CT value of each microRNA, calculate the expression abundance of each microRNA by-Δ Δ Ct method again, calculation formula is-Δ Δ Ct=-{ (Sample Cttarget-Sample CtmiR-16)-(Control Cttarget-ControlCtmiR-16) };
(4) choose suitable internal control gene, pick out the purpose differential gene.
2. the method for microRNA expression level is characterized in that in the detection ight soil according to claim 1, and total microRNAs is got in the extracting of described ight soil extraction agent box.
3. the method for microRNA expression level is characterized in that in the detection ight soil according to claim 1, selects suitable internal control gene with Genorm software.
4. a system of selection that is used to detect the internal control gene of carcinoma of the pancreas is characterized in that, mainly may further comprise the steps:
(1) stool sample with the Pancreas cancer patients of collecting extracts with ight soil extraction agent box, extracts the concentration of back with NanoDrop evaluation RNA;
(2) will identify that good RNA carries out reverse transcription and generates cDNA, by the expression of Real-time RT-PCR testing goal microRNA;
(3) obtain the CT value of each microRNA, calculate the expression abundance of each microRNA by-Δ Δ Ct method again, calculation formula is-Δ Δ Ct=-(Ct
Target microRNAs-Ct
MiR-16);
(4) choose suitable internal control gene.
5. the system of selection that is used to detect the internal control gene of carcinoma of the pancreas according to claim 4, it is characterized in that step (1) is: specimen collection places ice chest immediately behind frozen pipe, and the Ultralow Temperature Freezer that was transferred to-80 ℃ in 6 hours is preserved; Need during the RNA extracting of ight soil from frozen pipe, take by weighing ight soil, extract total microRNAs, extract the concentration of back with NanoDrop evaluation RNA with ight soil extraction agent box.
6. the system of selection that is used to detect the internal control gene of carcinoma of the pancreas according to claim 4, it is characterized in that, select suitable internal control gene with Genorm software: the expression amount of setting a certain house-keeping gene Ct value reckling in the different samples in Excel is 1, and the relative expression quantity of other samples and above-mentioned house-keeping gene then is 2
-Δ Ct, wherein, each sample Ct value of Δ Ct=-minimum Ct value; With the relative expression quantity data importing Genorm program that obtains; Utilize this program to calculate the mean value of genetic expression stability, the expression stability of house-keeping gene is sorted, be worth more for a short time, express stablely more, and judge the suitableeest number of house-keeping gene by the pairing variance analysis of house-keeping gene normalization factor.
7. according to each described system of selection that is used to detect the internal control gene of carcinoma of the pancreas of claim 4-6, it is characterized in that described internal control gene is miR-16.
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| CN107075578A (en) * | 2014-09-16 | 2017-08-18 | 东丽株式会社 | The comparative analysis method and apparatus of miRNA expression quantity |
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| CN101233243A (en) * | 2005-08-10 | 2008-07-30 | 财团法人浜松科学技术研究振兴会 | Method of detecting large bowel cancer marker |
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Cited By (3)
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| CN107075578A (en) * | 2014-09-16 | 2017-08-18 | 东丽株式会社 | The comparative analysis method and apparatus of miRNA expression quantity |
| CN107075578B (en) * | 2014-09-16 | 2021-06-01 | 东丽株式会社 | method and device for comparative analysis of miRNA expression level |
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Application publication date: 20110525 |