CN101818202A - Method for screening new internal reference molecules suitable for cervical tissue micro RNA real-time fluorescence quantitative PCR research - Google Patents
Method for screening new internal reference molecules suitable for cervical tissue micro RNA real-time fluorescence quantitative PCR research Download PDFInfo
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Abstract
The invention discloses a method for screening new internal reference molecules suitable for cervical tissue micro RNA real-time fluorescence quantitative PCR research. The method comprises the following technical steps: screening human Micro RNA probes by using a Micro RNA chip, and selecting Micro RNAs with constant normal tissue expression and high expression abundance of cervical cancer and cervix as candidate internal references; selecting a normal cervical epithelial tissue and a cancerous cervical tissue as clinical cervical tissue specimens respectively, and verifying the specimens by using fluorescence quantitative PCR; and analyzing data by using two special internal reference analysis software comprising geNorm software and Norm Finder so as to discover the internal reference molecules suitable for cervical tissue Micro RNA research. The internal reference molecules screened and verified by using the method of the invention can avoid possible differences of different cervical tissue specimens on the yield, quality and reverse transcription efficiency of the RNA, well correct and standardize the data for real-time fluorescence quantitative PCR detection, and improve the accuracy and the reliability of the search.
Description
Technical field
The present invention relates to a kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research.
Background technology
Cervical cancer is second largest malignant tumour of women that sickness rate is only second to mammary cancer, a large amount of epidemiologic datas show, it is the prerequisite of cervical cancer and precancerous lesion morbidity thereof that high-risk human papillomavirus (HPV) infects, but single HPV infects the vicious transformation that is not sufficient to cause epithelium of cervix uteri.Microrna (micro RNA, be called for short miRNA) be the little RNA of non-coding that the endogenous length of organism is about 20-23 Nucleotide, by on post-transcriptional level, expression of gene being carried out negative regulation, cause the degraded of mRNA or translation to suppress with the complementary pairing of said target mrna.Up to the present, reported that several thousand kinds of miRNA are present in the many cells eukaryotes such as animal, plant, fungi high conservative in the evolution.
Many studies have shown that, miRNA can be in order to the mark cancer, for example, the research of Ohio State Univ-Columbus USA is by analyzing 540 parts in the cancer cells sample located from lung, uterine neck, stomach, prostate gland, colon and pancreas etc., found the entity cancer miRNA signal that the most of miRNA by overexpression forms.And for tumor suppression and the oncogene of proteins encoded, the target of these miRNA differential expression effects can a large amount of enrichments, and this explanation miRNA plays an important role in the pathogenesis of cancer mechanism of noumenal tumour.Point out it very likely in HPV causes the generation evolution of cervical cancer, to bring into play keying action.Some MicroRNA abnormal expression in cervical cancer has been found in research in recent years, and therefore utilizing experimental biology and the bioinformatics method couple MicroRNA relevant with cervical cancer to study is one of focus of current research.
Understand MicroRNA role in gene regulating, a very crucial method be exactly rapidly, detection by quantitative MicroRNA expression of gene exactly.Sophisticated MicroRNA generally has only twenties bases, usually the difference of having only one or two base between the consanguinity MicroRNA, and the expression level of most MicroRNA in cell is all lower, these characteristics are given traditional blot hybridization (northern blot), and conventional meanses such as biochip technology (microarray) and RT-PCR have brought great difficulty and challenge.Along with PolyA RT-PCR technology, the especially development of stem-loopRT-PCR technology, real-time fluorescence quantitative PCR (real-time PCR) has become the main method of present MicroRNA detection by quantitative, this method is not only highly sensitive, the sample consumption is few, can detect low abundance target MicroRNA, the quantitative linearity wide ranges; And can accurately distinguish sequence height homologous MicroRNA in the same family, can accurately detect the expression level of the different micro RNA that have only a base difference.Suitable internal control gene is proofreaied and correct and stdn data but the accuracy that real-time fluorescence quantitative PCR detects depends on screening to a great extent, thus the difference of avoiding different specimens on output, quality and the reverse transcription efficient of RNA, may exist.
To studies confirm that of mRNA, the so-called constant expression of any internal control gene all was under the cell of certain type or empirical factor effect the constant of scope to be arranged in the past.In the cell of other types or the empirical factor effect is next changes, use internal control gene blindly, may make the fine difference of genetic expression be difficult to find on the one hand, may cause mistake even opposite conclusion on the other hand.The expression level of MicroRNA in cell is low only to account for 0.01% of total RNA, and its expression has bigger tissue specificity, just seems particularly important so select suitable confidential reference items that stdn is carried out in the expression of purpose MicroRNA.The molecule that is used as MicroRNA QRT-PCR confidential reference items at present in the document mainly contains let-7a, MicroRNA-103a, MicroRNA-16, MicroRNA-191, MicroRNA-26b etc. and microRNA (RNU48,5S), it selects confidential reference items also different because of the difference of research organization of institute.
Up to the present, to many reference used reference molecules (RNU48,5S, let-7a) in other tumours blindly in the quantitative examination of the relevant specificity micro RNA of cervical lesions, but all probe into to its homoeostasis of in cervical tissue, expressing and as the suitability of confidential reference items.
Summary of the invention
Goal of the invention of the present invention, defective on the research effect that is to use at confidential reference items in the quantitative examination of specificity MicroRNA in the existing cervical tissue has proposed a kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research; Screening of utilization the inventive method and the reference molecules that confirms, the difference that can avoid the Gene in Different Cervical Tissues sample on output, quality and the reverse transcription efficient of RNA, may exist, better the data that real-time fluorescence quantitative PCR is detected are proofreaied and correct and stdn, improve the accuracy and the reliability of research.
For achieving the above object, the technical solution used in the present invention is:
A kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research the steps include:
(1) utilizes the MicroRNA chip that human MicroRNA probe is screened, select in cervical cancer and the uterine neck normal tissue expression is constant and express the high MicroRNA of abundance, as candidate's confidential reference items;
(2) choose normal cervical epithelial tissue and canceration cervical tissue respectively as clinical cervical tissue sample, verify with quantitative fluorescent PCR;
(3) the confidential reference items analysis software of utilization geNorm software and two special uses of NormFinder carries out data analysis, thereby finds the reference molecules applicable to cervical tissue MicroRNA research.
Further, in the described step (2), used sample is the tissue of biopsy under the clinical vaginal mirror.
Further, in the some parts of samples of gathering by step (2), every part of sample divides two parts, aly preserve through the rearmounted liquid nitrogen of liquid nitrogen flash freezer, another part after formaldehyde fixed with the embedding of stone vinegar.
Further, in the described step (2), be used for MicroRNA chip inspection screening reference molecules and candidate's reference molecules is carried out the checking of real-time fluorescence quantitative PCR through the liquid nitrogen cryopreservation tissue.
Further, in the described step (2), the tissue after the embedding of stone vinegar is used to carry out histology and confirms with checking whether pathological change to take place.
Beneficial effect of the present invention is:
The present invention is directed to the defective on the research effect that confidential reference items use in the quantitative examination that has specificity MicroRNA in the cervical tissue now, proposed a kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research; Screening of utilization the inventive method and the reference molecules that confirms, the difference that can avoid the Gene in Different Cervical Tissues sample on output, quality and the reverse transcription efficient of RNA, may exist, better the data that real-time fluorescence quantitative PCR is detected are proofreaied and correct and stdn, improve the accuracy and the reliability of research.
Description of drawings
Fig. 1 is total electrophoretic data plot of RNA in the screening step of the present invention;
Fig. 2 is in the screening step of the present invention, the quality inspection figure of MiRNA;
Fig. 3 is that the difference MicroRNA in normal cervical tissues and the cervical cancer tissues expresses spectrogram;
Fig. 4 is miRNA RT-PCR product size location electrophoresis result figure;
Fig. 5 is that MicroRNA RT-PCR product sequence is determined---confidential reference items U6 and has-mir-23a sequencing result figure;
Fig. 6 is MicroRNA real-time PCR primer amplification typical curve and solubility curve figure;
To be each candidate's reference molecules normally reach differential expression data plot in the cervical cancer tissues at uterine neck to Fig. 7;
Fig. 8 carries out geNorm software analytical results figure to candidate's internal control gene.
Embodiment
Below in conjunction with accompanying drawing the present invention is further explained and illustrates:
Shown in Fig. 1-8:
The present invention selects uterine neck normal epithelial tissue and each 30 example of cervical cancer tissues for use, the volunteer that the normal cervical epithelial source is collected in Hospital for Gynaecology and Obstetrics Attached to Medical Hospital of Zhejiang to carry out the cervical disease examination, cervical cancer tissues derives from the untreated cervical cancer patient (squama cancer 23 examples, adenosquamous carcinoma 4 examples, gland cancer 3 examples) of initial diagnosis.All samples are the tissue of biopsy under the vaginoscope, and every part of sample divides two parts, and are a through the rearmounted liquid nitrogen preservation of liquid nitrogen flash freezer, stone vinegar embedding after another part 4% formaldehyde fixed.The liquid nitrogen cryopreservation tissue is used for Micro RNA chip inspection screening reference molecules and candidate's reference molecules is carried out the checking of real-time fluorescence quantitative PCR.The section of stone vinegar is used for carrying out histology and confirms that healthy tissues does not have pathological change, and the cancer composition is greater than 80% in the tumor tissues.Clinical stages,, cell differentiation divided high, the low differentiation of neutralization by cellular form with reference to FIGO (FIGO) in 2009.I phases 22 example, II phases 6 example, above 2 examples of II phase in the 30 routine cervical cancers, high differentiation 5 examples, middle differentiation 21 examples, low differentiation 4 examples.
The picked at random uterine neck normally reaches each 6 example of cancerous tissue sample respectively from the liquid nitrogen cryopreservation tissue, delivers to U.S. LC science company after total RNA extracts successfully and carries out Micro RNA separation, quality inspection and chip (LC science microarray) scanning detection and chip data analysis work.Chip results is selected candidate's reference molecules by following principle: (1) normally reaches in the cancerous tissue all Micro RNA of high expression level at all uterine neck; (2) carry out statistical study by the z-score and the variation coefficient (CV), normally reach at uterine neck and all express constant Micro RNA in the cancerous tissue; (3) in each Micro RNA family, select a most representative Micro RNA.The molecule that is used as Micro RNA QRT-PCR confidential reference items at present mainly contains: let-7a, MicroRNA-103, MicroRNA-16, MicroRNA-191, MicroRNA-26b etc. and microRNA (RNU48,5S), the reference molecules of selecting for use in the cervical tissue Micro RNA QRT-PCR research has: RNU48,5S, let-7a.Merge chip data and literature search result, low molecule is expressed in rejecting in cervical tissue, finally choose the candidate confidential reference items of following molecule as cervical tissue Micro RNA quantitative examination: MicroRNA23a, MicroRNA200c, MicroRNA26a, MicroRNA1979, let7a, MicroRNA191, MicroRNA103, RNU48 and 5S.
Obtain the sequence of the ripe body of each MicroRNA from miRBase database (http://MicroRNA.sanger.ac.uk), utilization lasergene corresponding specific stem-loop RT of each MicroRNA of 6.0 software designs and real-timePCR primer, the specificity of primer all confirms through blast search.Get remaining liquid nitrogen cryopreservation tissue sample (normal group 24 examples, tumor group 24 examples), carry out the detection of RNA extraction, quality inspection, stem-loop method RT and quantitative fluorescent PCR (real-timePCR) respectively, capable TA clone of the product of PCR and order-checking are to determine its specificity subsequently.Use the confidential reference items analysis software of geNorm software and two special uses of NormFinder to carry out data analysis at last, thereby find reference molecules applicable to cervical tissue MicroRNA research.
Concrete experimental procedure and result are as follows:
1. the collection of sample and grouping
1). the source of sample: cervical cancer tissues 30 examples are the untreated patient of initial diagnosis, wherein squama cancer 23 examples, adenosquamous carcinoma 4 examples, gland cancer 3 examples; FIGO is I phases 22 example, II phases 6 example, above 2 examples of II phase by stages; Well-differentiated carcinoma 5 examples, middle differentiation cancer 21 examples, poor differentiated carcinoma 4 examples.The uterine neck normal epithelial is organized 30 examples, all from coming from through all negative volunteer of cervical disease examination.
2). fresh cervical tissue is obtained in biopsy under the vaginoscope, and every part of sample divides two parts, and is a through the rearmounted liquid nitrogen preservation of liquid nitrogen flash freezer, stone vinegar embedding after another part 4% formaldehyde fixed.Stone vinegar specimen embedding is made into the section of 4 μ m, carries out HE dyeing and immunohistochemical methods two-step process dyeing respectively, determines that further no pathology changes in the normal cervical tissues, cancerous tissue in the tumor tissues 〉=80% and histological type thereof.
2.MicroRNA chip detection and interpretation of result:
1). fresh cervical tissue 12 examples of preserving in the picked at random liquid nitrogen (cervical cancer 6 examples, normal cervix 6 examples), extract total RNA, carry out Micro RNA separation, quality inspection and chip (LC science microarray) scanning and detect.
Total RNA and the Micro RNA that meets Micro RNA chip detection standard extracts, quality inspection (seeing Fig. 1,2).
2) detected result of .MicroR NA chip and data analysis
Normal cervix and cervical cancer examination go out MicroRNA31 (see figure 3) of differential expression, express consistent MicroRNA, after just sorting by average expression amount, carry out statistical study, filter out candidate's confidential reference items Micro RNA molecule (seeing Table 1) with the z-score and the variation coefficient (CV).
Z-score=X-μ/σ (X is meant that certain MicroRNA is in the average signal value of all sample expression amounts in the chip, and μ is the mean number of X, and σ is that certain MicroRNA is at all sample expression amount standard errors).
Table 1: average signal value, CV and the z-score value of candidate's confidential reference items MicroRNA molecule
Mean????cv??????z-score??Rank
hsa-let-7a????32,952??0.1880??4.1891???1
hsa-let-7c????29,572??0.2161??4.6267???2
hsa-let-7f????25,201??0.2254??4.4368???3
hsa-let-7b????24,785??0.2335??4.2827???4
hsa-miR-26a???24,089??0.2346??4.2618???5
hsa-let-7d????22,682??0.2506??3.9907???6
hsa-miR-23b???20,679??0.2528??3.9557???7
hsa-miR-23a???18,568??0.2674??3.7401???8
hsa-miR-200c??10,225??0.2877??3.4754???9
hsa-let-7i????8,224???0.2910??3.4368???10
hsa-let-7g????9,020???0.3086??3.2407???11
hsa-miR-197???920,095?0.3255??3.0718???12
3. can be used as the molecule of Micro RNA QRT-PCR confidential reference items at present
The molecule that is used as Micro RNA QRT-PCR confidential reference items at present mainly contains: let-7a, MicroRNA-103, MicroRNA-16, MicroRNA-191, MicroRNA-26b etc. and microRNA (RNU48,5S), the reference molecules of selecting for use in the cervical tissue miRNA QRT-PCR research has: RNU48,5S, let-7a.
4. the real-time PCR of candidate's reference molecules detects:
Merge chip and literature search result and finally choose the candidate confidential reference items of following molecule: MicroRNA23a, MicroRNA200c, MicroRNA26a, MicroRNA1979, let7a, MicroRNA191, MicroRNA103a, RNU48 and 5S as cervical tissue Micro RNA quantitative examination.
1). obtain the sequence of the ripe body of each Micro RNA from miRBase database (http://MicroRNA.sanger.ac.uk)
2). utilization lasergene corresponding specific stem-loop RT of each Micro RNA of 6.0 software designs and real-timePCR primer, the specificity of primer all confirms through blast search.Each primer sees the following form 2.
Table 2: corresponding specific stem-loop RT of candidate MicroRNA and real-timePCR primer
| Gene (base Because of) | ??Ac??ces??sio??n??nu??mb ??er | ??RNA??speci ??es | ??specific??stem-loop?RT ??primer[5’-3’] | ??specific??PCR??forward??primer??sequence[5 ??’-3’] | ??miRNA??reverse??PCR ??primer | ??corr??elat??ion??coe??ffici??ents ??(R2) | ??PCR??Ampli??ficati??on??effici??ency( ??％) |
| ??miRNAL ??et-7a | ??MI??MA??T00??000 ??62 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CAACTA | ??GCCGCTG??AGGTAGTA ??GGTTGTA | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??5 | ??98 |
| ??miRNA-2 ??3a | ??MI??MA??T00??000 ??78 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CGGAAAT | ??AGCGGAT??CACATTGC ??CAGGG | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??6 | ??99 |
| ??miRNA-2 ??6a | ??MI??MA??T00??000 ??82 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CAGCCTA | ??CGCAGTTC??AAGTAATC ??CAGG | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??5 | ??103 |
| Gene (base Because of) | ??Ac??ces??sio??n??nu??mb ??er | ??RNA??speci ??es | ??specific??stem-loop?RT ??primer[5’-3’] | ??specific??PCR??forward??primer??sequence[5 ??’-3’] | ??miRNA??reverse??PCR ??primer | ??corr??elat??ion??coe??ffici??ents ??(R2) | ??PCR??Ampli??ficati??on??effici??ency( ??％) |
| ??miRNA-2 ??00c | ??MI??MA??T00??006 ??17 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CTCCATC | ??GAGCGTA??ATACTGCC ??GGGTA | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??7 | ??102 |
| ??miRNA-1 ??979 | ??MI??MA??T00??094 ??54 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CTAGTCA | ??CCGGACT??CCCACTG ??CTTCAC | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??5 | ??100 |
| ??miRNA-1 ??91 | ??MI??MA??T00??004 ??40 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CCAGCTG | ??GCAGCCA??ACGGAAT ??CCCAAA | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??6 | ??98 |
| Gene (base Because of) | ??Ac??ces??sio??n??nu??mb ??er | ??RNA??speci ??es | ??specific??stem-loop?RT ??primer[5’-3’] | ??specific??PCR??forward??primer??sequence[5 ??’-3’] | ??miRNA??reverse??PCR ??primer | ??corr??elat??ion??coe??ffici??ents ??(R2) | ??PCR??Ampli??ficati??on??effici??ency( ??％) |
| ??miRNA-1 ??03 | ??MI??MA??T00??001??01 | ??Micro ??RNA | ??GTCGTATCCAG??TGCAGGGTCC??GAGGTATTCGC??ACTGGATACGA ??CTCATAG | ??CGGAGAG??CAGCATTG ??TACAG | ??GTGCAG??GGTCCG ??AGGT | ??0.99 ??9 | ??108 |
| ??RNU48 | ??NC??_00??001 ??5.9 | ??snoR ??NA | ??AACGCTTCAC ??GAATTTGCGT | ??CTCGCTTC??GGCAGCA ??CA | ??AACGCTT??CACGAAT ??TTGCGT | ??0.99 ??9 | ??99 |
| ??5s | ??NR??023??379 ??.1 | ??snoR ??NA | ??random?6mers | ??TACGGCC??ATACCAC ??CCTGA | ??GGCGGT??CTCCCAT ??CCAA | ??0.99 ??9 | ??100 |
3). candidate's reference molecules carries out real-time PCR and detects (ABI 7900)
Choose the other 48 routine fresh cervical tissue samples of preserving in the liquid nitrogen (normal 24 examples, tumour 24 examples), extract RNA, quality inspection and MicroRNA stem-loop method RT, RNU48 carries out RT with its special primer, and 5S carries out RT with random primer.
MicroRNA stem-loop method RT system (10 μ l):
total?RNA??????????????????????????????500pg
10X?RT?Buffer(TaKaRa?Biotechnology)????2.0μl
2.5mM?dNTPs(TaKaRa)????????????????????1.0μl
5x?RT?Primer(invitrogen)???????????????1.0μl
40U/mL?RNase?Inhibitor?Protein(TaKaRa)?0.2μl
100U/ul?wt-MMLV-RT(Ambion)?????????????0.5μ
RNA-free?water????????????????up?to????10μl
The RT loop parameter is as follows:
16℃×30min→42℃×30min→85℃×5min.
Get above-mentioned RT product, use Applied Biosystems 7900HT to carry out the detection of quantitative fluorescent PCR (real-timePCR) respectively.Electrophoresis by adding solubility curve, product and TA cloning and sequencing are to determine the specificity of PCR product.
A.real-timePCR reaction system (20 μ l):
cDNA?????????????????????????????????2μl
SYBR?green?I(TaKaRa?Biotechnology)???10μl
50X?ROX?Standard?????????????????????0.4μl
10x?PCR?forward?primer(invitrogen)???0.5μl
10x?PCR?reverse?primer(invitrogen)???0.5μl
The PCR loop parameter is as follows:
95 ℃ of pre-sex change 10`min
↓
(95 ℃ * 10s, 60 ℃ * 30s) * 40 circulation
↓
72 ℃ are extended 10min
B.Micro RNA RT-PCR product size location---electrophoresis result shows that the size of product at 60-70bp, is the purpose segment of required amplification, sees Fig. 4 for details.
C.Micro RNA RT-PCR product sequence is definite---and confidential reference items U6 and has-mir-23a the sequencing analysis coincidence rate of display sequence as a result are 100%.See Fig. 5: MicroRNA RT-PCR product sequence is definite---confidential reference items U6 and has-mir-23a sequencing result.
D.Micro RNA real-time PCR primer amplification efficient and specificity are definite---and typical curve and solubility curve detect.Typical curve shows that amplification efficiency reaches 100%, and solubility curve shows that the specificity of product is good.
4) candidate's reference molecules real-time PCR detected result:
Each candidate's reference molecules expression levels in cervical tissue is all used the Ct value representation, its uterine neck normally reach in the cervical cancer tissues differential expression as shown in Figure 7, its the mobility scale of maximum, minimum Ct value and the expression thereof expressed in a organized way as shown in table 3.
Table 3: the maximum that each candidate's reference molecules is expressed in cervical tissue, minimum Ct value and mobility scale thereof
| ??Gene?symbol | ??Ct?Range | ??Ct?Min | ??Ct?Max | ??Mean?Ct±sd |
| ??MicroRNA200c | ??5.17 | ??16.07 | ??21.23 | ??18.61±1.31 |
| ??MicroRNA26a | ??4.82 | ??15.98 | ??20.80 | ??17.86±1.10 |
| ??MicroRNA23a | ??2.04 | ??18.96 | ??21.00 | ??19.82±0.51 |
| ??MicroRNA1979 | ??3.88 | ??16.55 | ??20.43 | ??18.37±0.91 |
| ??MicroRNA191 | ??2.37 | ??18.52 | ??20.90 | ??19.58±0.53 |
| ??MicroRNA103a | ??4.05 | ??21.82 | ??25.87 | ??22.98±0.79 |
| ??let-7a | ??3.78 | ??21.88 | ??25.66 | ??23.67±0.92 |
| ??RNU48 | ??2.38 | ??7.43 | ??9.81 | ??8.60±2.38 |
| ??5S?rRNA | ??4.86 | ??10.20 | ??15.06 | ??12.53±1.12 |
5) utilization geNorm software and NormFinder confidential reference items analysis software carry out data analysis
The analytical results of NormFinder:
The analysis of NormFinder shows that MicroRNA23a is the most stable confidential reference items of cervical tissue MicroRNA research, the confidential reference items combination of 5s and MicroRNA191 the best.
2) geNorm software analytical results:
This program with the ratio in twos of a certain internal control gene and other internal control gene expression levels after logarithmic transformation, calculate the mean value M of its average difference as the genetic expression stability, expression stability to all candidate's internal control genes sorts, and normalization factor is matched variance analysis judge the suitableeest number of required internal control gene.
Candidate's internal control gene sorts by the M value, and uncomfortable cooperation confidential reference items are thought in M＞1.5, and M value novel Benq more is stable more because of expressing.That geNorm analysis demonstration is the most stable is MicroRNA 23a and MicroRNA 191 (as shown in Figure 8).
Screen the reference molecules The selection result that draws through screening method of the present invention:
Micro RNA 23a is the suitable confidential reference items in the quantitative examination of the relevant specificity Micro RNA of a kind of newfound cervical lesions, confidential reference items (RNU48,5S, let-7a) commonly used before its homoeostasis all is better than.
Below by test method concrete screening process of the present invention is described::
For example: the differential expression that detects MicroRNA 375 in uterine neck healthy tissues and the cervical cancer tissues with MicroRNA 23a as confidential reference items with the real-time fluorescence quantitative PCR method.
The experimental implementation process:
1. the extraction of cervical tissue MicroRNA, quality inspection:
1) per 50～100mg tissue carries out after the cracking Trizol lysate being changed in the EP pipe to tissue with 1ml Trizol reagent, places 5 minutes under room temperature 15～30C;
2) in above-mentioned EP pipe, the amount that adds the 0.2ml chloroform according to every 1ml Trizol adds chloroform, covers EP pipe lid, firmly shakes in hand 15 seconds, and at room temperature (15 ℃～30 ℃) were placed after 2～3 minutes, centrifugal 15 minutes of 12000g (2 ℃～8 ℃);
3) get the upper strata water and place new EP pipe, the amount that adds the 0.5ml Virahol according to every 1ml TRIZOL adds Virahol, and at room temperature (15 ℃～30 ℃) were placed 10 minutes, centrifugal 10 minutes of 12000g (2 ℃～8 ℃);
4) abandon supernatant, add 1ml 75% ethanol according to every 1ml TRIZOL and wash, vortex mixed, centrifugal 5 minutes of 7500g (2 ℃～8 ℃) abandons supernatant;
5) allow sedimentary RNA seasoning at room temperature;
6) utilize spectrophotometer, agarose gel electrophoresis to detect the RNA concentration and the purity of sample.(qualified standard is OD 260/280 ≈ 2 (1.9 to 2.2), 28S/18S ratio 〉=1.7)
2.MicroRNA 23a and MicroRNA 375 specific stem-loop RT and real-time fluorescence quantitative PCR primer design:
1) obtains the sequence of the ripe body of each MicroRNA from miRBase database (http://MicroRNA.sanger.ac.uk)
2) utilization lasergene corresponding specific stem-loop RT of each MicroRNA of 6.0 software designs and real-timePCR primer, the specificity of primer all confirms through blast search.
3. MicroRNA 23a in the cervical tissue and MicroRNA375 are carried out reverse transcription with the stem-loop method respectively:
MicroRNA stem-loop method RT system (10 μ l):
total?RNA??????????????????????????????500pg
10X?RT?Buffer(TaKaRa?Biotechnology)????2.01μl
2.5mM?dNTPs(TaKaRa)????????????????????1.0μl
5x?RT?Primer(invitrogen)???????????????1.0μl
40U/mL?RNase?Inhibitor?Protein(TaKaRa)?0.2μl
100U/ul?wt-MMLV-RT(Ambion)?????????????0.5μ
RNA-free?water????up?to????10μl
The RT loop parameter is as follows:
16℃×30min→42℃×30min→85℃×5min.
Instrument: (48-well GeneAmp PCR System 9700)
4. get above-mentioned RT product, carry out the detection of quantitative fluorescent PCR (real-timePCR) respectively:
Real-timePCR reaction system (20 μ l):
cDNA????????????????????????????????2μl
SYBR?greenI(TaKaRa?Biotechnology)???10μl
50X?ROX?Standard????????????????????0.4μl
10x?PCR?forward?primer(invitrogen)??0.5μl
10x?PCR?reverse?primer(invitrogen)??0.5μl
The PCR loop parameter is as follows:
95 ℃ of pre-sex change 10`min
↓
(95 ℃ * 10s, 60 ℃ * 30s) * 40 circulation
↓
72 ℃ are extended 10min
Instrument: Applied Biosystems 7900HT
5. data analysis:
Utilizing SPSS16.0 statistics software, is that confidential reference items utilize MicroRNA 375 expression difference between 2 (Delta CT) methods statistical study uterine neck normal group and cervical cancer group with MicroRNA 23a.
It should be noted that embodiment only is to explanation of the present invention, it should be considered as restriction and qualification, therefore, adopt flesh and blood of the present invention and only make local change, must fall within the scope of protection of the present invention technical scheme of the present invention.
Claims (5)
1. screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research is characterized in that the step of this screening method is:
(1) utilizes the MicroRNA chip that human MicroRNA probe is screened, select in cervical cancer and the uterine neck normal tissue expression is constant and express the high MicroRNA of abundance, as candidate's confidential reference items;
(2) choose normal cervical epithelial tissue and canceration cervical tissue respectively as clinical cervical tissue sample, verify with quantitative fluorescent PCR
(3) the confidential reference items analysis software of utilization geNorm software and two special uses of NormFinder carries out data analysis, thereby finds the reference molecules applicable to cervical tissue MicroRNA research.
2. a kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research according to claim 1, it is characterized in that: in the described step (2), used sample is the tissue of biopsy under the clinical vaginal mirror.
3. a kind of screening method that is applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research according to claim 1, it is characterized in that: in the some parts of samples by step (2) collection, every part of sample divides two parts, a preserve through the rearmounted liquid nitrogen of liquid nitrogen flash freezer, another part after formaldehyde fixed with the embedding of stone vinegar.
4. according to claim 1 or 3 described a kind of screening methods that are applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research, it is characterized in that: in the described step (2), be used for MicroRNA chip inspection screening reference molecules and candidate's reference molecules is carried out the checking of real-time fluorescence quantitative PCR through the liquid nitrogen cryopreservation tissue.
5. according to claim 1 or 3 described a kind of screening methods that are applicable to the new reference molecules of cervical tissue micro RNA real-time fluorescence quantitative PCR research, it is characterized in that: in the described step (2), the tissue after the embedding of stone vinegar carries out histology and confirms with checking whether pathological change to take place.
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