CN109182329A - A kind of application for the RNA extraction method of paraffin-embedded tissue sample and its in high-flux sequence - Google Patents
A kind of application for the RNA extraction method of paraffin-embedded tissue sample and its in high-flux sequence Download PDFInfo
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Abstract
The present invention relates to tissue samples process fields, specifically, design a kind of RNA extraction method for paraffin-embedded tissue sample and its application in high-flux sequence, first interrupt the dimethyl bridged linkage of protein and nucleic acid in the tissue samples, the nucleic acid in the protein and nucleic acid crosslinking is released again, isolate nucleic acid and remove DNA to get.The method has RNA recovery rate height, quality height, shortens time, simple operation and other advantages.
Description
Technical field
The present invention relates to tissue samples process field, in particular to a kind of for paraffin-embedded tissue sample
RNA extraction method and its application in high-flux sequence.
Background technique
The preservation of tissue samples generallys use the fixation of formalin and the mode of paraffin embedding.In this way, former
The morphological feature of tissue samples is completely kept down.With the development of molecular biology technology, more and more points
Sub- diagnosis business all uses the tissue samples of formalin fixed paraffin embedding (FFPE).But FFPE will affect tissue point
Quality from obtained DNA and RNA.By the tissue of fixed embedding, fragmentation will occur for nucleic acid；Formaldehyde can be sent out with nucleic acid
Biochemical reaction and generate it is corresponding change, the crosslinking etc. including protein and other biological molecule.In the mistake that sample is fixed
Self-dissolving occurs for Cheng Zhong, histocyte and other cell components, leads to the fracture of RNA.Hot environment in embedding process and
Prolonged storage will all aggravate the fracture of RNA.Meanwhile during being sliced storage, the RNA on surface is exposed to through peroxide
Change, in addition the experiment flows such as slice dyeing, all will irreversibly influence the RNA mass extracted.
The traditional RNA extraction method of FFPE tissue samples is based primarily upon the extracting method of Proteinase K, utilizes cell cracking
Liquid lytic cell, protease K digesting albumen obtain the RNA of initial gross separation.It is isolated in the environment of lysate with high salt
RNA by washing, removes the extra protein and other impurities adsorbed on splitter, finally using low in conjunction with splitter
The RNA combined on the buffer elution magnetic bead of salt.In traditional extracting method, protease K digesting is committed step.But egg
The white enzyme K processing time is long, generally 15min~3h, even in this way, the tissue by enzymic digestion can not also illustrate to be to fill
Divide digestion.Although and Proteinase K can digest the protein ingredient in corsslinking molecular to discharge albumen and nucleic acid cross-linked structure
In RNA, but cannot destroy those do not contain peptide bond dimethyl bridged linkages.Therefore, FFPE tissue samples are traditional
RNA extraction method receives the limitation of Proteinase K self-characteristic.
Fixed paraffin embedding (FFPE) tissue samples of formalin are important biomedical research material.Most of feelings
Under condition, FFPE sample contains a large amount of and clinically relevant information, but therefrom to extract RNA is very difficult one
Work.RNA molecule not only with protein it occur frequently that crosslinking in chemical structure, but also can because the degradation of RNA molecule and
Influence the quality and quantity of extraction product RNA.In addition, frequently encountering sample during the extraction process since tissue samples are usually limited
The situation of this amount deficiency.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of RNA extraction method for paraffin-embedded tissue sample, the methods
, quality height, shortening time, simple operation and other advantages high with RNA recovery rate.
It is another aspect of the invention to provide application of the said extracted method in high-flux sequence, and high pass can be improved
Measure the precision of sequence.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of RNA extraction method for paraffin-embedded tissue sample first interrupts protein and core in the tissue samples
The dimethyl bridged linkage of acid, then the nucleic acid in the protein and nucleic acid crosslinking is released, it isolates nucleic acid and removes
DNA to get.
The RNA extraction method is used to prepare described by application of the above-mentioned RNA extraction method in high-flux sequence
The RNA template of high-flux sequence.
Compared with prior art, the invention has the benefit that
(1) extracting method of the invention solves the problems, such as albumen and RNA crosslinking to influence RNA extraction, improves
The quality and yield that RNA is extracted, so as to preferably do rna expression analysis using FFPE sample.
(2) RNA extraction method of the invention, the method before comparing improve about 10 times in RNA extraction efficiency,
RT-qPCR detection method display sensitivity also improves 2~3 circulations, high-flux sequence resulting RNA mass as the result is shown
Also than using the extracted RNA mass of the prior art more excellent or without difference.
(3) shorten extraction time: largely shorten Proteinase K handles the time using time and DNAse.
Specific embodiment
When using traditional RNA extraction method, it is low usually to encounter extraction efficiency, and it is insufficient to extract sample initial amount
Situation.Since conventional method uses Proteinase K, after enzymic digestion, still remaining many microscopic tissues
Fragment illustrates that tissue is not digested adequately.In order to overcome this bottleneck, we use following methods:
A method of RNA being extracted from the tissue samples of the fixed paraffin embedding of formalin, first interrupts the tissue sample
The dimethyl bridged linkage of protein and nucleic acid in this, then the nucleic acid in the protein and nucleic acid crosslinking is released, point
Separate out nucleic acid and remove DNA to get.
In some embodiments, described to interrupt to use ultrasound to be interrupted.
Ultrasound interrupts, and is directly to carry out fragmentation by way of physics to tissue, reduce Proteinase K to tissue
Digestion time.It is under the microscope or it can be seen that big even if having digested a period of time when using enzymic digestion tissue samples
The tissue block of piece, but interrupted by ultrasound, the use of Proteinase K, efficiency can be cooperated first by tissue fragmentation as far as possible
It is higher, therefore also save the plenty of time.
In some embodiments, the condition that the ultrasound interrupts are as follows: in the condition that ultrasonic power is 160~190W
200~400s of lower processing.
The ultrasonic frequency can also for 170,175,180,185W etc. (result is almost the same within the scope of this)；It is described
Time can also be (result are almost the same within the scope of this) such as 250s, 300s, 350s.
In some embodiments, the condition that the ultrasound interrupts further includes that temperature is 16~20 DEG C, service factor 8
~12%, incident power peak value is set as 160~190 watts, and every triggering recycles 150~250 times, handles the time 200~400 seconds, instrument
18~22 DEG C of device temperature.
Incident power peak value is the instantaneous ultrasonic power acted on sample；Service factor, that is, ultrasonic wave acts on sample
The time of product accounts for the percentage of period；Every triggering circulation is that ultrasonic wave acts on transfer of ultrasonic energy in sample
Number；Time, that is, duration is handled, the time of sample is operated.
In some embodiments, the method for the release is to use protein described in protease digestion.
In some embodiments, the condition of the digestion are as follows: 15~20min is handled at 45~55 DEG C.
The condition of the digestion can be with are as follows: handles 18min at 45 DEG C, handles 20min at 50 DEG C, at 55 DEG C
Handle (result are almost the same within the scope of this) such as 15min.
In some embodiments, in the system of the digestion, the volumetric concentration of the protease is 3%~5%.
The volumetric concentration of the protease can also be 3.5%, 4%, 4.5% etc. (result is almost the same within the scope of this).
In some embodiments, protease includes Proteinase K.
It in some embodiments, further include the inactivation of secondary digestion and the protease after the digestion.
In some embodiments, the method for the inactivation of the secondary digestion and the protease is included at 78~82 DEG C
Handle 15~20min.
On the one hand, Proteinase K can be made to inactivate for 78~82 DEG C, make experiment will not because of Proteinase K activity and influence
Subsequent extraction, on the other hand, paraffin section group can generate protein and the crosslinking of RNA when being woven in fixed using formalin,
At first such structure by protease K digesting a part, can further be destroyed under this range temperature environment later, have
Conducive to the extraction of RNA.
The method of the inactivation of the secondary digestion and the protease can be with are as follows: 17min is handled at 78 DEG C, 80
15min is handled at DEG C, is handled at 82 DEG C (result are almost the same within the scope of this) such as 20min.
It in some embodiments, further include carrying out the tissue samples at slice and dewaxing before described interrupt
Reason.
In some embodiments, after described interrupt, the nucleic acid released is the core dissolved after described interrupt
Acid.
The temperature can also be 80 DEG C equal (result is almost the same within the scope of 78~82 DEG C).
In some embodiments, the method for isolating nucleic acid is to be extracted using dehydrated alcohol.
In some embodiments, the method for the removal DNA includes handling 30~40min using DNAse.
The time of the processing can be also (result are almost the same within the scope of this) such as 32min, 35min, 38min.
In some embodiments, the volumetric concentration of the DNAse is 6%~9%.
The volumetric concentration of the DNAse can also be 6.6%, 7%, 8% etc. (result is almost the same within the scope of this).
In some embodiments, described further includes purifying when isolating nucleic acid and removing DNA, and the purifying is affine
Purifying.
Purification process can be used different kits and carry out.
In some embodiments, the extraction RNA is carried out in no RNAase system.
Throughout the extraction process, used container, solution etc. are without RNAase.
In some embodiments, the length of the tissue samples is less than 10mm；Preferably 3~8mm.
Tissue samples are too long to be unfavorable for postorder ultrasound and interrupts.
It is another aspect of the invention to provide application of the said extracted method in high-flux sequence, and the RNA is mentioned
Method is taken to be used to prepare the RNA template of the high-flux sequence.
It can guarantee the integrality and amount of original sample, reduce pollution and error, improve the precision of high-flux sequence.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument
Person, being can be with conventional products that are commercially available.
Embodiment 1
It is placed in being put in centrifuge tube by the tissue samples of slice and dewaxing on ice.If sample is longer than 10mm, in advance
It is first cut to less than 10mm.Sample usesE220 ultrasound interrupts instrument and is interrupted, water level setting 10, and cooling temperature is
18 DEG C, service factor 10%, incident power peak value is set as 175 watts, and every triggering recycles 200 times, handles the time 300 seconds, instrument
20 DEG C of temperature.The part of dissolution is transferred in clean centrifuge tube.
The protease K digesting buffer of 100 μ L and protease (the Recover All of 4 μ L is addedTM Total Nucleic
Acid Isolation Kit for FFPE), sufficient vortex mixes and infiltrates sample sufficiently.
Sample is incubated 15 minutes at 50 DEG C, until sample mixtures clarification, is then transferred at 80 DEG C and incubates 15 points
Clock.
Arrange in pairs or groups Recover AllTMTotal Nucleic Acid Isolation Kit for FFPE kit carries out
The extraction of RNA: the separation mixed liquor of 395 μ L of configuration, 100% alcohol comprising 120 μ L separation additive and 275 μ L.To newly it match
The separation mixed liquor and sample mixtures set are sufficiently mixed and are transferred to and collect in column.10,000g centrifugations 30 seconds, discard centrifugation
Collection liquid.The cleaning solution 1 of 700 μ L is added to collecting in column, 10,000g centrifugations 30 seconds discard and liquid is collected by centrifugation.500 μ L are added
To collecting in column, 10,000g centrifugations 30 seconds discard and liquid are collected by centrifugation cleaning solution 2.10,000g centrifugations 30 seconds, discard collection column
Residual liquid.The DNase mixture of 60 μ L is added in collecting pipe, the 10X DNase buffer including 6 μ L, the DNase of 4 μ L
And 50 μ L nuclease-free water.It incubates 30 minutes at room temperature.Repeat the cleaning step of above cleaning solution 1 and cleaning solution 2.It will
It collects column to be placed in new collecting pipe, 60 μ L nuclease-free waters is added, stand 1 minute at room temperature, rapid centrifugation sample, elution
RNA.It extracts obtained RNA and is put in -80 DEG C of preservations before analysis.RNA mass and quantity are by Agilent High Sensitivity
RNA Screen Tape Kit detection, and software analysis is carried out by 4200 Tape Statin Software.
Embodiment 2
It is placed in being put in centrifuge tube by the tissue samples of slice and dewaxing on ice.If sample is longer than 10mm, in advance
It is first cut to less than 10mm.Sample usesE220 ultrasound interrupts instrument and is interrupted, water level setting 10, and cooling temperature is
18 DEG C, service factor 10%, incident power peak value is set as 160 watts, and every triggering recycles 200 times, handles the time 200 seconds, instrument
20 DEG C of temperature.The part of dissolution is transferred in clean centrifuge tube.
The protease K digesting buffer of 100 μ L and protease (the Recover All of 5 μ L is addedTM Total Nucleic
Acid Isolation Kit for FFPE), sufficient vortex mixes and infiltrates sample sufficiently.
Sample is incubated 15 minutes at 45 DEG C, until sample mixtures clarification, is then transferred at 78 DEG C and incubates 15 points
Clock.
Arrange in pairs or groups Recover AllTMTotal Nucleic Acid Isolation Kit for FFPE kit carries out
The extraction of RNA: the separation mixed liquor of 395 μ L of configuration, 100% alcohol comprising 120 μ L separation additive and 275 μ L.To newly it match
The separation mixed liquor and sample mixtures set are sufficiently mixed and are transferred to and collect in column.10,000g centrifugations 30 seconds, discard centrifugation
Collection liquid.The cleaning solution 1 of 700 μ L is added to collecting in column, 10,000g centrifugations 30 seconds discard and liquid is collected by centrifugation.500 μ L are added
To collecting in column, 10,000g centrifugations 30 seconds discard and liquid are collected by centrifugation cleaning solution 2.10,000g centrifugations 30 seconds, discard collection column
Residual liquid.The DNase mixture of 60 μ L is added in collecting pipe, the 10X DNase buffer including 5 μ L, the DNase of 5 μ L
And 50 μ L nuclease-free water.It incubates 35 minutes at room temperature.Repeat the cleaning step of above cleaning solution 1 and cleaning solution 2.It will
It collects column to be placed in new collecting pipe, 60 μ L nuclease-free waters is added, stand 1 minute at room temperature, rapid centrifugation sample, elution
RNA.It extracts obtained RNA and is put in -80 DEG C of preservations before analysis.RNA mass and quantity are by Agilent High Sensitivity
RNA Screen Tape Kit detection, and software analysis is carried out by 4200 Tape Statin Software.
Embodiment 3
It is placed in being put in centrifuge tube by the tissue samples of slice and dewaxing on ice.If sample is longer than 10mm, in advance
It is first cut to less than 10mm.Sample usesE220 ultrasound interrupts instrument and is interrupted, water level setting 10, and cooling temperature is
18 DEG C, service factor 10%, incident power peak value is set as 190 watts, and every triggering recycles 200 times, handles the time 400 seconds, instrument
20 DEG C of temperature.The part of dissolution is transferred in clean centrifuge tube.
The protease K digesting buffer of 100 μ L and protease (the Recover All of 3 μ L is addedTM Total Nucleic
Acid Isolation Kit for FFPE), sufficient vortex mixes and infiltrates sample sufficiently.
Sample is incubated 20 minutes at 55 DEG C, until sample mixtures clarification, is then transferred at 82 DEG C and incubates 20 points
Clock.
Arrange in pairs or groups Recover AllTMTotal Nucleic Acid Isolation Kit for FFPE kit carries out
The extraction of RNA: the separation mixed liquor of 395 μ L of configuration, 100% alcohol comprising 120 μ L separation additive and 275 μ L.To newly it match
The separation mixed liquor and sample mixtures set are sufficiently mixed and are transferred to and collect in column.10,000g centrifugations 30 seconds, discard centrifugation
Collection liquid.The cleaning solution 1 of 700 μ L is added to collecting in column, 10,000g centrifugations 30 seconds discard and liquid is collected by centrifugation.500 μ L are added
To collecting in column, 10,000g centrifugations 30 seconds discard and liquid are collected by centrifugation cleaning solution 2.10,000g centrifugations 30 seconds, discard collection column
Residual liquid.The DNase mixture of 60 μ L is added in collecting pipe, the 10X DNase buffer including 5 μ L, the DNase of 5 μ L
And 50 μ L nuclease-free water.It incubates 35 minutes at room temperature.Repeat the cleaning step of above cleaning solution 1 and cleaning solution 2.It will
It collects column to be placed in new collecting pipe, 60 μ L nuclease-free waters is added, stand 1 minute at room temperature, rapid centrifugation sample, elution
RNA.It extracts obtained RNA and is put in -80 DEG C of preservations before analysis.RNA mass and quantity are by Agilent High Sensitivity
RNA Screen Tape Kit detection, and software analysis is carried out by 4200 Tape Statin Software.
Experimental example 1
The extracted amount of RNA.
The setting of comparative example.Based on embodiment 1, the setting of example is compared:
Comparative example 1: without ultrasonication；
Comparative example 2: without ultrasonication, and using protease K digesting 3h, handles 60min using DNAse.
Remaining is same as Example 1, and the paraffin section including selection derives from identical tissue site, similarly mentions
Take sample size.The RNA extracted amount of tumor tissues sample one and tumor tissues sample two is detected respectively, and each method is done puts down three times
Row experiment.It the results are shown in Table 1 and table 2.
1 Different Extraction Method of table detects the comparing result of RNA extracted amount in tumor tissues sample one
2 Different Extraction Method of table detects the comparing result of RNA extracted amount in tumor tissues sample two
In table 1 as can be seen that extraction side of the tumor tissues sample one using extracting method of the invention than comparative example 1 and 2
The recovery rate of method, RNA increases.It is same to extract sample size, the extraction of RNA when control group sample uses the method for the present invention
Amount is increased to 74ng (average value of embodiment 1) by the about 9.0ng of comparative example, improves 8.1 times；Use comparative example 1 and 2
Extracting method has no significant effect the recovery rate of RNA, illustrates when without using ultrasonication, even if extend the digestion of albumen
Between and RNAse the processing time, it is still little to extraction rate impact.
From Table 2, it can be seen that the extracted amount of RNA is by comparative example when tumor tissues sample two is using method of the invention
About 8.5ng be increased to 106.3ng (average value of embodiment 1), improve 12.5 times；Use the extraction side of comparative example 1 and 2
Method has no significant effect the recovery rate of RNA in tumor tissues sample, illustrates when without using ultrasonication, even if extending albumen
Digestion time and RNAse the processing time, it is still little to extraction rate impact.
Based on the above results, the extracted amount of the average RNA obtained using extracting method of the invention is than using comparative example
Method increases about 10.3 times.
Experimental example 2
The extraction effect of RT-qPCR detection tumor sample RNA.
Comparative example is arranged in the method for reference experiment example 1.
By analyzing the expression quantity of conservative gene, to confirm the extraction effect of the method for the present invention and comparative example method.It selects
Expressing gene be glyceraldehyde phosphate dehydrogenase (GAPDH).
Use the RNA of extraction as template, iScriptTMCDNA Synthesis Kit (BioRad) carries out reverse transcription
PCR reaction.After traditional method extracts RNA, carry out cDNA synthesis and reacted with quantitative fluorescent PCR, each sample need using
The RNA of 1.0-2.5ng.In order to guarantee that experiment under equal conditions carries out, all experiments are usedPreAmp
Master Mix (Applied BiosystemsTM) is expanded in advance.Pre- amplification carries out 14 PCR with reference to kit specification
Circulation.RT-qPCR uses KAPA PROBE FAST qPCR Kit Master Mix Universal reagents (Kapa
Biosystems).The cDNA template of 2.5 μ L is added in each reaction, and total volume is 10 μ L.Use 7500 (Applied of ABI
Biosystems RT-qPCR experiment) is carried out.
Experimental result is shown in Table 3.
The extraction effect comparing result for the RNA that 3 Different Extraction Method of table is extracted
From table 3 it can be seen that when the RNA template of equivalent participates in reaction, the RNA that is obtained using extracting method of the invention
As template, the Cq value in RT-qPCR is less about two compared to the Cq value that the RNA participation reaction that comparative example is extracted obtains and follows
Ring illustrates that extracting method of the invention obtains RNA when participating in reaction, and sensitivity is higher under similarity condition；Use comparative example 1
Extracting method with 2 has no significant effect the Cq value of RNA, illustrates when without using ultrasonication, even if extending disappearing for albumen
The processing time for changing time and RNAse influences extraction effect still little.
Experimental example 3
The extraction effect of high-flux sequence detection RNA.
Use SMARTer Stranded Total RNA-seq Kit-Pico Input Mammalian from
Clontech/Takara Bio USA prepares the library RNA and removal rRNA.With reference to kit specification, using 10ng
RNA template carry out high-flux sequence.
The method of RNA extraction method and comparative example 2 of the template from the embodiment of the present invention 1.Sequencing is sequenced using both-end
Method, sequencing reading length 150bp, use 550 microarray dataset of Illumina NextSeq, 500/550 v2 of NextSeq examination
Agent box, detailed step refer to kit specification.
The Reads that all sequencings generate is compared with genomic DNA.The Reads of 48.35%-78.41% can be with
It is compared with genomic DNA.Two methods do not have too big difference in the result of the Reads of generation.By Reads and outer aobvious
The DNA of subregion is compared, and the method for the RNA Reads comparing ratio 1 obtained that method of the invention is extracted is high
Out 1.55%.In general, two kinds of extracting methods do not have much influence high-flux sequence result, and the two result is similar.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；
Although present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that:
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of technologies
Feature is equivalently replaced；And these are modified or replaceed, the present invention that it does not separate the essence of the corresponding technical solution is each to be implemented
The range of example technical solution.
Claims (10)
1. a kind of RNA extraction method for paraffin-embedded tissue sample, which is characterized in that first interrupt egg in the tissue samples
The dimethyl bridged linkage of white matter and nucleic acid, then the nucleic acid in the protein and nucleic acid crosslinking is released, isolate core
Acid and remove DNA to get.
2. the method according to claim 1, wherein described interrupt to use ultrasound to be interrupted；Preferably, institute
State the condition that ultrasound interrupts are as follows: 200~400s is handled under conditions of ultrasonic power is 160~190W.
3. the method according to claim 1, wherein the method for the release is to use egg described in protease digestion
White matter；
Preferably, the condition of the digestion are as follows: 15~20min is handled at 45~55 DEG C.
4. according to the method described in claim 3, it is characterized in that, in the system of the digestion, the volume of the protease
Concentration is 3%~5%；
Preferably, protease includes Proteinase K.
5. according to the method described in claim 4, it is characterized in that, further including secondary digestion and the protease after the digestion
Inactivation；
Preferably, the method for the inactivation of the secondary digestion and the protease is included in 15~20min of processing at 78~82 DEG C.
6. the method according to claim 1, wherein before described interrupt, further include by the tissue samples into
Row slice and dewaxing treatment.
7. the method according to claim 1, wherein the method for isolating nucleic acid be using dehydrated alcohol into
Row extracts.
8. the method according to claim 1, wherein the method for the removal DNA includes using DNAse processing 30
~40min；Preferably, the volumetric concentration of the DNAse is 6%~9%；
Preferably, described further includes purifying when isolating nucleic acid and removing DNA, and the purifying is affinity purification.
9. described in any item methods according to claim 1~9, which is characterized in that the extraction RNA is in no RNAase system
It carries out；
Preferably, the length of the tissue samples is less than 10mm；Preferably 3~8mm.
10. application of the RNA extraction method according to any one of claims 1 to 9 in high-flux sequence, which is characterized in that will
The RNA extraction method is used to prepare the RNA template of the high-flux sequence.
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| CN110684830A (en) * | 2019-10-11 | 2020-01-14 | 深圳吉因加医学检验实验室 | RNA analysis method for paraffin section tissue |
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