CN102181431A

CN102181431A - Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues

Info

Publication number: CN102181431A
Application number: CN 201110059474
Authority: CN
Inventors: 崔世英; 丁艳芳; 赵瑾瑶
Original assignee: Dalian Medical University
Current assignee: Dalian Medical University
Priority date: 2011-03-11
Filing date: 2011-03-11
Publication date: 2011-09-14
Anticipated expiration: 2031-03-11
Also published as: CN102181431B

Abstract

The invention discloses a method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues, which is an improvement method provided on the basis of a kit. The method comprises the following steps: deparaffinating, digesting, extracting and purifying. By adopting the method, the RNA in a tissue sample stored by being embedded by paraffin can be extracted, wherein, the longest storage time reaches up to 6 years; the RNA is analyzed by a nucleic acid trace tester and an internationally recognized Agilent 2100bioanalyzer; the extraction concentration is 211-3116ng/mu l, and the extraction purity is A260/280:1.75-2.04, which are all very ideal; and the expression research of microarray high-abundance genes of qRT-PCR and miRNA in mRNA and miRNA level can be carried out, which provides an experimental basis for developing a large amount of research on formalin fixed paraffin-embedded (FFPE) genes, and has wide application value.

Description

From paraffin-embedded tissue, extract the method for total RNA

Technical field

The present invention relates to the RNA extracting method, the extracting method of total RNA in the especially long-time formalin fixed paraffin-embedded tissue.

Background technology

Formalin fixed paraffin-embedded tissue (FFPE) is the method for the most frequently used preservation pathological tissue of medical institutions, because fresh specimens is difficult to obtain, for carrying out the noumenal tumour molecular biology research, can only from paraffin-embedded tissue, carry out gene when existing case is carried out retrospective study and extract, so the paraffin-embedded tissue the abundantest precious resources that just become source.The method of extracting DNA from paraffin-embedded tissue is ripe, but the research at DNA can not accurately reflect the change that takes place in the disease related gene transcription sometimes to a certain extent, therefore from pathology archives sample, extract capacity and the RNA that can effectively increase significant to genome functions and polymorphism research.But from paraffin-embedded tissue, extract RNA for a long time and have some difficulties always: on the one hand, compare with DNA, RNA is very unstable, be subjected to the influence of factors such as envrionment temperature, potential of hydrogen easily, especially ubiquitous RNA enzyme, in fixing and embedding treatment process, be easy to make the RNA molecule, be not suitable for being further used for molecular biology research by chemically modified or degraded; On the other hand, caused tissue protein one RNA's of fixing agent is highly cross-linked, makes that RNA more is difficult to extract.Recent years, many progress appear in this respect both at home and abroad, but extracting method is different, and also there are very big difference in the output of gained RNA and amplification efficiency.The extracting method of reporting in the document, mostly be the Trizol cracking process, protease digestion method or test kit extraction method, but concentration and the purity of the RNA that extracts are undesirable, degraded is serious, the small fragment RNA amplification can only be carried out, more the research of real-time quantitative PCR (q RT-PCR) and gene chip (microarray) can not be further well carried out.And the prior art application at object, promptly the FFPE sample mostly is the sample in 1 year, this also brings very big restriction for the application of clinical study.Therefore, need the more effective method that can be applicable to the RNA extraction of the FFPE sample that the longer time preserves of research and development.

Summary of the invention

The method that the object of the present invention is to provide a kind of RNA that can be used for the long-time FFPE sample of preserving to extract, the present invention is at test kit (RecoverAll ^TMTotal Nucleic Acid Isolation Kit, Ambion company product, article No. AM1975) the basis on proposed improve one's methods, this method comprises the steps:

1. dewaxing: pending paraffin-embedded tissue is cut into the tissue that thickness is 20 μ m, getting 4 tissue places in the aseptic 1.5ml centrifuge tube, add 1ml dimethylbenzene, whirlpool concussion mixing, 50 ℃ of water-baths 3 minutes, under the room temperature condition, centrifugal 4 minutes of 14000rpm, abandon supernatant, repetitive operation 1～3 time; Add the 1ml dehydrated alcohol, mixing, under the room temperature condition, centrifugal 2～4 minutes of 14000rpm abandons supernatant, repetitive operation 2 times, gained sample chamber warm air drying 15～30 minutes;

2. digestion: 1. add 400ul digestion damping fluid and 4ul proteolytic enzyme in the gained sample to step, 55 ℃ of water-baths 30～150 minutes;

3. extract: add the 1100ul dehydrated alcohol in the 480ul extraction agent, add in the centrifuge tube, blowing and beating repeatedly with suction nozzle does not have agglomeratingly up to tissue, and 90% tissue crumble is till the cloud that is white in color; Prepare the collection tube of a band filter post, add the 700ul mixed solution, centrifugal 30 seconds of 9000rpm adds remaining liq again, and 9000rpm is centrifugal 30 seconds again, abandons filtrate; Add 700ul washing lotion 1, centrifugal 30 seconds of 9000rpm adds 500ul washing lotion 2/3, and centrifugal 30 seconds of 9000rpm abandons filtrate, and 9000rpm is centrifugal 30 seconds again, abandons filtrate;

4. purifying: 95 ℃ of water-bath preheating nuclease free water in advance, prepare the DNase mixed solution: 6ul 10xDNA enzyme buffer liquid, 4ul DNA enzyme, 50ul nuclease free water; Add 60ul DNA enzyme mixed solution to filter post central authorities, room temperature left standstill 30 minutes, added 700ul washing lotion 1 room temperature and left standstill 30～60 seconds, centrifugal 30 seconds of 9000rpm, abandon filtrate, added 500ul washing lotion 2/3 9000rpm centrifugal 30 seconds, abandon filtrate, add 500ul washing lotion 2/3 again, centrifugal 60 seconds of 9000rpm abandons filtrate; With new collection tube, add the nuclease free water of the preheating of 60ul, room temperature left standstill 2～3 minutes, and centrifugal 1 minute of 12000rpm collects once repeatedly.

Adopt method of the present invention, can extract the shelf time and reach RNA in the tissue sample that the paraffin embedding in 6 years preserves most, and the carrying out of success follow-up internationally recognized molecular Biological Detection.And mostly the existing research of association area is to be less than at preservation period the sample in 1 year.All there is not bibliographical information both at home and abroad: extracted total RNA in the paraffin-embedded tissue of formalin fixed in 2～6 years in preservation, carry out q RT-PCR and the microarray of mRNA and miRNA.Analyze through nucleic acid trace test instrument and internationally recognized Agilent 2100bioanalyzer, the concentration of extracting is at 211-3116ng/ul, dna purity A260/280:1.75-2.04, all very desirable, can further carry out the high abundance expression of gene research of the microarray of the qRT-PCR of mRNA and miRNA level and miRNA, this provides experiment basis for the gene studies of carrying out lot of F FPE, has wide application value.

Description of drawings

Accompanying drawing 6 width of cloth of the present invention,

Fig. 1 is the RNA agarose electrophoresis result of the formalin fixed paraffin-embedded tissue of the human ovarian cancer extracted of method of the present invention, and numeral is represented the sample label respectively among the figure.

Fig. 2 is the RNA mass analysis collection of illustrative plates of the formalin fixed paraffin-embedded tissue of the human ovarian cancer extracted of method of the present invention, wherein:

Fig. 2 (A) is the analytical results collection of illustrative plates of standard substance 1, Ladder:RNA scope: 218; RNA concentration: 150ng/ul;

Fig. 2 (B) and Fig. 2 (C) are respectively the analytical results collection of illustrative plates of No. 21, No. 12 samples, reference standard 1;

Fig. 2 (D) is the analytical results collection of illustrative plates of standard substance 2, Ladder:RNA scope: 223.7; RNA concentration: 150ng/ul;

Fig. 2 (E) is the analytical results collection of illustrative plates of No. 14 samples, reference standard 2;

Fig. 2 (F) is the analytical results collection of illustrative plates of standard substance 3, Ladder:RNA scope: 281.3; RNA concentration: 150ng/ul;

Fig. 2 (G), 2 (H), 2 (I), 2 (J) and 2 (K) are respectively the analytical results collection of illustrative plates of 22,24,29,34 and No. 40 samples, reference standard 3.

Accompanying drawing 3 is that the RNA of the formalin fixed paraffin-embedded tissue of the human ovarian cancer extracted of method of the present invention is used for miRNA gene chip (Microarray) experimental result spectrogram.

Accompanying drawing 4 is the confidence level collection of illustrative plates of the microarray result in the different ovarian cancer samples of being expressed in of miRNA.

To be method of the present invention 1 terms of extracting be used for mRNA q RT-PCR detected result spectrogram with the RNA of the formalin fixed paraffin-embedded tissue of interior human ovarian cancer with accompanying drawing 5, wherein:

Fig. 5 (A) is the amplification curve of internal control gene GAPDH,

Fig. 5 (B) is the amplification curve of goal gene STMN1,

Fig. 5 (C) is the melt curve analysis of internal control gene GAPDH,

Fig. 5 (D) is the melt curve analysis of goal gene STMN1.

To be method of the present invention 6 terms of extracting be used for miRNA q RT-PCR detected result spectrogram with the RNA of the formalin fixed paraffin-embedded tissue of interior human ovarian cancer with accompanying drawing 6, wherein:

Fig. 6 (A) is the amplification curve of confidential reference items miRNA U6,

Fig. 6 (B) is the amplification curve of purpose miRNA.

Embodiment

The nucleic acid extraction kit of U.S. Ambion company, Recoverall ^TMTotal Nucleic AcidIsolation Kit (article No. AM1975) is the tool reagent box that those skilled in the art use always.The present inventor finds under study for action, and this test kit extracts and has certain limitation being used for paraffin-embedded tissue sample RNA, and when being applied to the long sample of preservation period, concentration and the purity of the RNA of extraction are not ideal enough.And, in the concrete application process of test kit,, the contriver also has many improvements but finding the method for this test kit, based on this, provide the RNA extracting method that comprises steps such as dewaxing, digestion, extraction and purifying of the present invention, specifically comprise the steps:

Above-mentioned steps 1. in, the operation that described employing dimethylbenzene dewaxes to pending paraffin-embedded tissue, promptly " pending paraffin-embedded tissue is cut into the tissue that thickness is 20 μ m, gets 4 tissue and place in the aseptic 1.5ml centrifuge tube, add 1ml dimethylbenzene; whirlpool concussion mixing; 50 ℃ of water-baths 3 minutes, under the room temperature condition, centrifugal 4 minutes of 14000rpm; abandon supernatant " can adjust number of times by paraffinicity per sample: paraffinicity is higher than at 50% o'clock, repetitive operation 3 times; Paraffinicity 30～50% o'clock, repetitive operation 2 times; Paraffinicity is lower than at 30% o'clock, operates 1 time.This step has been carried out refinement to the dewaxing number of times on the basis of kit method, be the more efficient and effective of sample.

Above-mentioned steps 1. in, behind dehydrated alcohol processing sample, available suction nozzle extrusion tissue, ethanol during exhaustion is organized as far as possible, handling gained sample 15～30 minutes required time of drying in next step operation under this improvement condition, the 45min more required than reagent cassette method is shorter, and has improved the exsiccant effect, and then shortens the time of next step digestion.

Above-mentioned steps 2. in, the consumption of digestion damping fluid, digestion temperature and digestion time all grope to improve through the contriver, improve the effect of digestion, especially at the storage life sample of length, improvement effect is especially obvious.

The equal comparison reagent kit of centrifugal revolution in this step reduces, can better protection filter post.

4. purifying: 95 ℃ of water-bath preheating nuclease free water, prepare the DNase mixed solution: 6ul 10x DNA enzyme buffer liquid, 4ul DNA enzyme, 50ul nuclease free water; Add 60ul DNA enzyme mixed solution to filter post central authorities, room temperature left standstill 30 minutes, added 700ul washing lotion 1 room temperature and left standstill 30～60 seconds, centrifugal 30 seconds of 9000rpm, abandon filtrate, added 500ul washing lotion 2/39000rpm centrifugal 30 seconds, abandon filtrate, add 500ul washing lotion 2/3 again, centrifugal 60 seconds of 9000rpm abandons filtrate; With new collection tube, add the nuclease free water of the preheating of 60ul, room temperature left standstill 2～3 minutes, and centrifugal 1 minute of 12000rpm collects once repeatedly.

Above-mentioned steps 4. in, 95 ℃ of water-bath preheating nuclease free water are the steps that do not have in the kit method, can increase the extraction concentration of RNA.Centrifugal revolution also decreases in this step, and protection filter post guarantees to filter effect better.

The Recoverall that reaches described in above-mentioned present method ^TMTotal Nucleic Acid Isolation Kit is available from U.S. Ambion company, article No.: AM1975.In the aforesaid method of the present invention: 2. described digestion damping fluid of step and proteolytic enzyme, the collection tube of the extraction agent that 3. step is addressed, band filter post and the 10x DNA enzyme buffer liquid that 4. step is addressed, the DNA enzyme, washing lotion 1 and washing lotion 2/3 are in the test kit and provide, and corresponding product is respectively in the pairing test kit:

Extraction agent: isolation additive;

10x DNA enzyme buffer liquid: 10xDNase buffer;

DNA enzyme: Dnase;

Washing lotion 1:wash1;

Washing lotion 2/3:wash2/3.

The described nuclease free water of the method for the invention described above, Thermo Scientific product, available from Hyclone, article No.: SH30538.01;

The dimethylbenzene of the invention described above (analytical pure, AR), available from the triumphant letter chemical industry in Tianjin company limited, lot identification mark on September 10th, 2010;

The dehydrated alcohol of the invention described above (analytical pure, AR), 10009218, available from Chemical Reagent Co., Ltd., Sinopharm Group, lot identification mark: 20091105.

Following embodiment is used to further specify the present invention.

Embodiment 1

Sample RNA extracts

The material of present embodiment is: the formalin fixed paraffin-embedded tissue of human ovarian cancer, and on behalf of tissue, 1M, 2M, 3M, 4M, 5M, 8M, 10M, 11M, 2Y, 3Y, 4Y, 5Y, 6Y in the present embodiment begin to be respectively 1 month to the shelf time that experimentizes from fixing embedding respectively, 2 months, 3 months, 4 months, 5 months, 8 months, 10 months, 11 months, 2 years, 3 years, 4 years, 5 years, 6 years.

21 of samples in 1 year: number consecutively is 1～21.

The sample in 2～6 years: 21: number consecutively is 22～42.

This numbering system also is applicable to other embodiment of this specification sheets.

From above-mentioned 42 samples, extract RNA with method of the present invention, through nucleic acid trace test instrument (brand: NanoVue Plus ^TM, GE Healthcare product, the U.S., model: 28923216) concentration of Ce Dinging and purity such as following table 1:

Table 1

Embodiment 2

The RNA agarose electrophoretic analysis.

Experimental technique and condition: (the Invitrogen product, article No.: inv-750024) clean, the point sample of selecting pore size to be fit to is combed, and vertical rack is at an end of hectograph with 0.1% DEPC water with electrophoresis chamber and sample comb.Take by weighing 1g agarose (Biowest agarose, GENE TECH company limited product, 111760), add 100ml1 * TAE (50x TAE, green skies biotechnology research institute product, article No.: ST716) in the electrophoretic buffer, the microwave oven heating makes the agarose dissolving evenly.Add 4 μ l EB (10mg/ml) (ethidium bromide, green skies biotechnology research institute product, article No.: ST059).Treat that gel is cooled to about 50 ℃, gel is poured into poured into gently on the running gel plate, remove bubble.After treating that gel solidifies, carefully take out the point sample comb.In electrophoresis chamber (DYCP-31F type, Beijing Liuyi Instrument Factory), add 1 * TAE electrophoretic buffer, offset plate is put into electrophoresis chamber, make electrophoretic buffer not have the glue face.6 * sample-loading buffer of adding 1/6 volume in the testing sample (6 * Loading Buffer, precious biotechnology (Dalian) company limited product, article No.: D604), and after the mixing, liquid-transfering gun point sample, the about 1ug of applied sample amount.120 volts of voltages, electrophoresis 30 minutes; In the gel imaging instrument, observe, take pictures.Experimental result as shown in Figure 1.

From detected result: 1 year clear with 18S and the 28S band of the RNA that interior sample extracts, the RNA less degradation; The sample in 2～6 years, though the RNA degraded increases, still as seen the part band is enough to be used in the relevant molecular biology experiment of follow-up miRNA.

Embodiment 3

The RNA mass analysis

Experimental technique and condition: adopt Agilent 2100 (Agilent) biological analyser (Agilent 2100bioanalyzer) that RNA integrity index is quantized fully, can measure the RNA sample more accurately, assess the RNA sample quality quantitatively, applied sample amount is 1ul only.

Experimental result as shown in Figure 2.As seen: result and agarose electrophoresis be basically identical as a result, used RNAladder RNA scope is between 218～281.3,1 year with interior No. 12 samples (Fig. 2 .C) and No. 14 samples (Fig. 2 .E) in the visible obviously peak shape of 18S and 28S place, illustrate that the RNA degraded is less, No. 21 samples (Fig. 2 .B) are though slightly degraded, the 18S peak disappears, but the 28S peak is clear; The RNA of sample extraction in 2～6 years, the part sample is (as No. 22, Fig. 2 .G) RNA of Ti Quing is in the also visible obviously peak shape of 18S and 28S place, the prompting degraded is less, though the part sample: No. 24 (Fig. 2 .H), No. 29 (Fig. 2 .I), No. 34 (Fig. 2 .J), the crest reach of No. 40 (Fig. 2 .K), the RNA degraded increases, but the molecular biology experiment that RNA quality of extracting and concentration are enough to carry out follow-up miRNA level.

Embodiment 4

MiRNA gene chip (Microarray) experiment

MiRNA in detection by quantitative tissue or the cell sample is the first step of miRNA functional study, helps people further to understand the relation of miRNA and disease development, for the early diagnosis of disease provides new thinking.This experiment mainly detects the expression of miRNA by chip technology and real-time quantitative PCR (Quantitative real-timePCR).

Experimental technique and condition:

Experiment material is the RNA that the sample in 2～6 years extracts, and sample is numbered 22～No. 42, and totally 21 samples are as embodiment 1.

Experimental technique:

1.miRNA chip preparation: the design and synthetic (the TaqManHuamn MicroRNA A+B Card v2.0 that prepare and finish special stem ring reverse transcriptase primer and quantification PCR primer by the Dana Farber of Harvard University tumour molecular diagnosis experimental center, Applied Biosystems, 768 primers, article No.: 4400238).

2.miRNA the collection of qRT-PCR check and analysis and chip image and data processing: No. 22～No. 42 sample rnas (all RNA adopt Agilent Bioanalyzer 2100 quantitatively to reach quality examination), use stem ring primer to carry out reverse transcription, (equipment: Applied Biosystems 7900HTFast Real-Time PCR System), the raw data that obtains uses RealTime statMiner 4.21Software to carry out Data Management Analysis in the qRT-PCR check and analysis.

Laboratory test results is shown in accompanying drawing 3 and accompanying drawing 4.

Accompanying drawing 3 shows: different miRNA genes are expressed pedigree in the ovarian cancer sample, are benchmark with zero among the figure, and the rising blue line is for raising, and the decline blue line is downward modulation (as Fig. 3 .A).Fig. 3 .B shows the miRNA gene of statistical significance and express pedigree in the ovarian cancer sample.Its ordinate zou shows the multiple that rises or descend.

Accompanying drawing 4 is depicted as the confidence level of the microarray result in the different ovarian cancer samples of being expressed in of miRNA, the different pattern detection results' of red representative degree of agreement is 100%, green is represented as 0, from then on figure can find out obviously that the credible result degree of the resulting microarray of total RNA that extracted the tissue sample from 2～6 years is very high, satisfactory for result.

Embodiment 5

Fluorescence real-time quantitative PCR (qRT-PCR) detects

(1), carries out the qRT-PCR of mRNA level with the sample (1,3,5,7,8,12, No. 14 samples) in a year.

Experimental technique and condition: experiment material: High Capacity cDNA RT kit, SYBR GREENPCR MASTER MIX, (American AB I company) uses SYBR GREEN I fluorescence dye method PCR, presses the operation of ABI specification sheets experiment condition.

Experiment key instrument: Agilent STRATAGENE Mx3000P quantitative real time PCR Instrument.

Experimental result is as shown in Figure 5:

Human?GAPDH：NCBI?Reference?Sequence：NM_002046.3

Human?STMN1(stathmin?1)：NCBI?Reference?Sequence：NM_005563.3

Experiment conclusion: adopt method of the present invention from 1 year total RNA, can use q RT-PCR method, amplify the product of mRNA goal gene effectively, carry out follow-up molecular biology research to extract the interior tissue sample.

(2) with 6 years with interior sample (22～No. 42), carry out the q RT-PCR of miRNA level.

Use the sophisticated Quantitative real-time RT-PCR technology expression of the purpose microRNA of detection by quantitative ovarian cancer tissue quickly and accurately, verify the experimental result of miRNA gene chip.

Experimental technique and condition: Taqman miRNA assay, U6miRNA, taqman PCR mastermix, Taqman microRNA RT Kit carry out RT and PCR operation all available from American AB I company according to the ABI specification sheets.

Experimental result as shown in Figure 6.As seen: the total RNA that adopts method of the present invention to extract the tissue sample from 2～6 years, can use q RT-PCR method, amplify the gene product of purpose miRNA effectively.

Claims

1. from paraffin-embedded tissue, extract the method for total RNA, comprise the steps:

1. dewaxing: pending paraffin-embedded tissue is cut into the tissue that thickness is 20 μ m, gets 4 tissue and place in the aseptic 1.5ml centrifuge tube, add 1ml dimethylbenzene, whirlpool concussion mixing, 50 ℃ of water-baths 3 minutes are under the room temperature condition, 14000rpm abandons supernatant, repetitive operation 1～3 time; Add the 1ml dehydrated alcohol, mixing, under the room temperature condition, centrifugal 2～4 minutes of 14000rpm abandons supernatant, repetitive operation 2 times, gained sample chamber warm air drying 15～30 minutes;

4. purifying: 95 ℃ of water-bath preheating nuclease free water in advance, prepare the DNase mixed solution: 6ul 10xDNA enzyme buffer liquid, 4ul DNA enzyme, 50ul nuclease free water; Add 60ul DNA enzyme mixed solution to filter post central authorities, room temperature left standstill 30 minutes, added 700ul washing lotion 1 room temperature and left standstill 30～60 seconds, centrifugal 30 seconds of 9000rpm, abandon filtrate, added 500ul washing lotion 2/39000rpm centrifugal 30 seconds, abandon filtrate, add 500ul washing lotion 2/3 again, centrifugal 60 seconds of 9000rpm abandons filtrate; With new collection tube, add the nuclease free water of the preheating of 60ul, room temperature left standstill 2～3 minutes, and centrifugal 1 minute of 12000rpm collects once repeatedly.

2. the described method of claim 1, it is characterized in that adopting during described step is 1. dimethylbenzene that the operation paraffinicity per sample that pending paraffin-embedded tissue dewaxes is adjusted number of times: paraffinicity is higher than at 50% o'clock, repetitive operation 3 times; Paraffinicity 30～50% o'clock, repetitive operation 2 times; Paraffinicity is lower than at 30% o'clock, operates 1 time.