CN109022418A - A kind of fast dewaxing method that FFPE sample nucleic acid extracts - Google Patents
A kind of fast dewaxing method that FFPE sample nucleic acid extracts Download PDFInfo
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Abstract
The invention discloses a kind of fast dewaxing methods that FFPE sample nucleic acid extracts, it is related to paraffin sample nucleic acid and extracts field, FFPE extracting method of the invention is incubated for by single temperature, using the oily phase reagent of dissolvable paraffin and the water phase reagent of cleavable tissue samples, realize simple and quick FFPE sample dewaxing, after this kind of reagent is added in the sample tube added with FFPE, it is incubated for 30 minutes to 2 hours under specific temperature conditions, the liquid of lower layer's aqueous layer is taken out with mechanical device or liquid-transfering device, which is that can be used for subsequent nucleic acid extraction operation.The present invention also provides a kind of kits that fast dewaxing is carried out using this method.The present invention carries out dewaxing and Tissue Lysis operation using single temperature, simplifies experiment flow, shortens whole dewaxing duration；Using specific reagent system, it can be achieved that comparatively ideal dewaxing and nucleic acid extraction effect.
Description
Technical field
The present invention relates to extracted from paraffin embedding sample nucleic acid more particularly to a kind of FFPE sample nucleic acid extract it is quick
Process for dewaxing.
Background technique
Formalin fixes paraffin embedding techniques (Formalin-Fixed and Paraffin-Embedded, FFPE)
A kind of tissue preservation techniques.This technology is first fixed with formalin in order to maintain Nuclear extract structure and then uses tissue
Solid paraffin embedding uses for making histodiagnosis or research after slice.
If to organize to carry out molecular biology research to FFPE, the extraction for carrying out DNA is sought to first.But fixative
Crosslinked action nucleic acid structure is damaged, the fixed extraction that nucleic acid can be interfered with paraffin embedding effect of formalin tissue inhibits
The activity of archaeal dna polymerase is to influence PCR amplification.In addition to this, paraffin hinders infiltration of the digestive juice to tissue, to inhibit egg
The contact of white enzyme K and albumen in tissue influence tissue digestion and DNA release.So in order to eliminate paraffin to DNA extraction and PCR
The adverse effect of amplification, it is necessary to guarantee not increasing exogenous PCR inhibiting factor, and reduce the premise of DNA additional injuries to the greatest extent
Under to tissue thoroughly dewaxed.
Applying the process for dewaxing in mature kit at present is mainly organic solvent lost-wax process, wherein most widely used
For the dimethylbenzene lost-wax process to dewax more thoroughly.1) dimethylbenzene process for dewaxing, which specifically includes that, to be immersed in FFPE in xylene solution
It fullys shake, is dissolved in paraffin in dimethylbenzene；2) xylene solution dissolved with paraffin is removed by way of centrifugation；3) exist
Dehydrated alcohol is added in the tissue precipitating of removal dimethylbenzene to carry out open texture structure, digestive juice is promoted to penetrate into and remove trace in tissue
The dimethylbenzene of amount；4) centrifugation removal ethanol solution, 5) digestion buffer and Proteinase K is added, by high-temperature process twice, often
Secondary each hour digests tissue, finally obtains postdigestive nucleic acid solution and carries out subsequent nucleic acid extraction operation.As current
Process for dewaxing most widely used, commercial kit is most, dimethylbenzene process for dewaxing still have more problem: firstly, two
Toluene is a kind of toxic organic reagent, unfavorable to the health of experimenter；Secondly, dimethylbenzene process for dewaxing needs to carry out multistep
Reagent addition and centrifugally operated, experimentation is cumbersome, dewaxing cleavage step at least time-consuming 3 hours.
In order to simplify the dewaxing process of paraffin-embedded tissue, there are also a kind of better simply silicone oil to crack two step lost-wax processes at present
[application number: 201310370788.0].It comprises the concrete steps that, avirulent wax cracking liquid and Tissue lysates is added to centrifugation
It is mixed in pipe and with the paraffin organization sample being sliced, after 60 DEG C and 90 DEG C are respectively incubated for 1 hour, the liquid in pipe can shared oil
Layer and two layers of water layer, from liquid is taken out in water layer come the sample after being dewaxed.But it since it is after 60 DEG C have been incubated for, needs to take
Sample tube out, thermal modules to be added place into after being warming up to 90 DEG C, make core too long to avoid solution crosslinking time caused by temperature-rise period
The excessive fragmentation of acid；And during taking out the water phase below oil reservoir, solidification of the upper oil phase as caused by temperature change etc.
Problem easily leads to and takes the problems such as liquid is difficult and liquid-taken amount is inaccurate or can not take out sample solution at all, while being easily introduced stone
Wax clast dewaxes cleavage step time-consuming at least two hour, in addition extraction step, which is entirely tested, needs or so 3 hours.
Existing dimethylbenzene process for dewaxing, there are toxicity for deparaffinization reagents, and slightly misoperation all can be right during the experiment
The personal safety of experimenter causes damages, and has certain risk when in use；Secondly, its need to carry out the addition of multistep reagent and from
Heart operation, experimentation is cumbersome, time-consuming.And silicone oil cracks the incubation time that two step lost-wax processes need at least two hour, needs
Two different temperature need to take out sample cell during temperature changes, each of which increases the whole dewaxing time at
Sheet and operation complexity, are unfavorable in conjunction with automation equipment.
Therefore, those skilled in the art be dedicated to developing a kind of increase operation convenience, reduce dewaxing heating time and
Harmful reagent is reduced to use and the process for dewaxing for realizing the novel FFPE nucleic acid extraction automated can be facilitated.
Summary of the invention
In view of the above drawbacks of the prior art, at present process for dewaxing when heated between, operation convenience and automation can
The technical issues of remaining unsolved in terms of row.Based on above-mentioned process for dewaxing basis, the present invention uses specific reagent system,
Simplify heating stepses early period, directlys adopt single temperature and be incubated for while realizing sample dewaxing and Tissue Lysis, substantially shorten dewaxing
The heating and cooling performance requirement to heating module is reduced while time, it is convenient in conjunction with relevant automatic device, realize fast
Fast efficient FFPE sample nucleic acid extracts.
To achieve the above object, the present invention provides a kind of fast dewaxing method that FFPE sample nucleic acid extracts, features
It is, comprising the following steps:
Step 1: FFPE sample and water phase reagent and oily phase reagent are sufficiently mixed, the first mixed liquor is formed；
Step 2: the first mixed liquor is heated to the arbitrary temp within the scope of 50-95 DEG C, sufficiently it is incubated at such a temperature
10min-72h forms the second separating liquid, and second separating liquid upper layer is oily phase, and lower layer is water phase；
Step 3: taking out lower layer's water phase, nucleic acid extraction is carried out；
The oil phase reagent is the oil-phase solution with immiscible, the dissolvable paraffin of water, and the water phase reagent is cracking FFPE
Sample with paraffin is immiscible, water-soluble aqueous phase solution, the water phase reagent includes pH buffer, surfactant and gold
Belong to chelating agent.
Further, first mixed liquor can be totally submerged FFPE sample.
Further, the oily phase reagent be in mineral oil, paraffin oil and silicone oil any one or it is a variety of.It is oily mutually to try
Agent can be the saturated hydrocarbons such as the mineral oil of dissolvable paraffin, paraffin oil, can also be liquid compatible with water it is immiscible, can dissolve stone
Any liquid deparaffinization reagents of wax.
Further, water phase reagent is used to crack tissue samples in FFPE, and type is unlimited, commercially available or be commonly used for
The water-soluble lysate for cracking biological tissue and cell is applicable in.Water phase reagent generally includes surfactant, as tween,
The amphoteric compounds such as span, Triton, lauryl sodium sulfate, neopelex, concentration≤10%.
Further, the water phase reagent further includes protein denaturant, such as rhodanate, isothiocyanate or perchloric acid
Salt etc., concentration≤6M.
Further, protease inhibitors, RNA enzyme etc. can be added in water phase reagent according to actual needs.
Further, the water phase reagent further includes Proteinase K.
Further, the ratio of water phase reagent and oily phase reagent can be completely dissolved paraffin without particular/special requirement with oily phase reagent,
Mixed solution, which can be totally submerged FFPE sample, to be advisable, and according to different FFPE sample sizes and thickness, the volume of water phase reagent is in 50 μ
L to 5mL, preferred dosage are 100 μ L to 500 μ L.The oily preferred dosage of phase reagent is in 50 μ L to 300 μ L, oily phase reagent and water
The ratio of phase reagent is in 1:4 to 2:1.
Further, heating schedule of the invention only needs a kind of temperature range, heating temperature between 50 DEG C -95 DEG C, compared with
Suitable temperature is 50 DEG C -70 DEG C, and more preferably temperature is 52 DEG C -65 DEG C；According to different tissues size and thickness (FFPE sample
Tissue is bigger, thickness is thicker, and the heating time needed can increase accordingly), the heating time that sample tissue is completely dissolved can increase
To 72 hours, it is contemplated that the timeliness problem of whole dewaxing cracking, appropriate time are 10min-24h, more preferably heating time
For 30min-3h.The demand of as needed and different water phase reagents, can increase subsequent solution crosslinking incubation time, and heating temperature exists
50 DEG C -95 DEG C, better suited temperature is 70 DEG C -95 DEG C, and more preferably temperature is 75 DEG C -93 DEG C；Heating time at 0-24 hours not
Deng, it is contemplated that timeliness problem, incubation relatively in due course between be 30min-2h, the more preferably time is 30min-1h.
Further, lower layer's aqueous phase solution can be taken out with automation equipment or liquid-transfering gun etc. in a heated condition, can also waited
Lower section aqueous phase solution is taken out to new centrifuge tube again after being cooled to room temperature completely, as having steam in pipe, of short duration centrifugation step can be increased
Suddenly, sample of nucleic acid is further taken out after so that liquid in pipe is arrived centrifugation bottom of the tube.The sample of nucleic acid can be tried with commercially available common paramagnetic particle method
Agent box, silicagel column method kit, automatic nucleic acid extract instrument etc. and carry out subsequent nucleic acid extraction.
The present invention also provides the fast dewaxing kit that a kind of FFPE sample nucleic acid extracts, which utilizes aforementioned side
The fast dewaxing of method progress FFPE sample.Kit includes aforementioned oily phase reagent and water phase reagent, according to size and thickness
Different temperature and incubation time is arranged in degree.
FFPE extracting method of the invention is incubated for by single temperature, using the oily phase reagent and cleavable of dissolvable paraffin
The water phase reagent of tissue samples realizes simple and quick FFPE sample dewaxing, this kind examination is added in the sample tube added with FFPE
It after agent, is incubated for 30 minutes to 2 hours under specific temperature conditions, takes out lower layer's water phase with mechanical device or liquid-transfering device and take out water
The aqueous phase liquid of phase layer, the aqueous phase liquid are the tissue samples solution that can be used for subsequent nucleic acid extraction operation.
Compared with prior art, the present invention having technical effect beneficial below:
1, heating stepses duration is reduced, using specific reagent system, it is only necessary to which FFPE can be realized in step temperature heating
Dewaxing and Tissue Lysis simplify dewaxing cleavage step without using toxic reagent, dewaxing is thoroughly and time-consuming short, simple and fast,
And extraction efficiency fully meets the application demands such as subsequent detection of nucleic acids；
2, overall process only needs same temperature, does not need to carry out heating and cooling operation, solution is needed in temperature-rise period by sample
The problem of this pipe moving-out device, simplify to the performance requirement of heating module and the operation complexity of self-reacting device, more conducively with
Automation equipment combines；
3, due to only needing same temperature, without the time that the time of heating module heating and cooling and subsequent high temperature are incubated for, greatly
Width shortens the whole dewaxing time, at the same when avoiding the proteopepsis reagents such as secondary plus Proteinase K caused by Aerosol Pollution.；
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is the FFPE nucleic acid extraction fast dewaxing schematic diagram of a preferred embodiment of the invention；
Fig. 2 is that nucleic acid concentration obtained by different FFPE sample nucleic acids extraction dewaxing treatment methods compares figure；
Fig. 3 is the 260/280 comparison figure that different FFPE sample incubation methods extract nucleic acid.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
In the accompanying drawings, the identical component of structure is indicated with same numbers label, everywhere the similar component of structure or function with
Like numeral label indicates.The size and thickness of each component shown in the drawings are to be arbitrarily shown, and there is no limit by the present invention
The size and thickness of each component.Apparent in order to make to illustrate, some places suitably exaggerate the thickness of component in attached drawing.
Water phase is added as shown in Figure 1, in the centrifuge tube 101 containing FFPE sample 102 in one embodiment of the present invention
Reagent and oily phase reagent mixture 103, after being put into the progress certain temperature heating of heating device 104, it is seen that liquid in centrifuge tube 101
Body is layered as oily phase layer 105 and aqueous layer 106, contains the nucleic acid sample cracked out from FFPE sample 102 in aqueous layer 106
This.At this point, lower layer's aqueous phase solution can be taken out with automation equipment or liquid-transfering gun etc. in a heated condition, it can also wait and be cooled to room temperature
Lower section aqueous phase solution is taken out to new centrifuge tube again afterwards completely, as having steam in pipe, of short duration centrifugation step can be increased, make intraluminal fluid
Body further takes out sample of nucleic acid 106 after arriving centrifugation bottom of the tube.The sample of nucleic acid 106 can with commercially available common paramagnetic particle method kit,
Silicagel column method kit, automatic nucleic acid extract instrument etc. and carry out subsequent nucleic acid extraction.Nucleic acid after present invention dewaxing cracking
Sample can be carried out subsequent with commercially available common paramagnetic particle method kit, silicagel column method kit, automatic nucleic acid extraction instrument etc.
Nucleic acid extraction.
Embodiment 1
By 200 μ L water phase reagent (10mM EDTA, 30mM Tris-HCl, 3M guanidinium isothiocyanate, 1% TritonX-
100), 20 μ L Proteinase Ks (5mg/mL) and 50 μ L oil phase reagents (silicone oil) be added the 1.5mL equipped with FFPE tissue samples 102 from
In heart pipe 101, of short duration centrifugation after concussion mixing obtains mixed liquor 103.
It is put into and has warmed up into 56 DEG C of heating device 104, be incubated for 1 hour under the conditions of 56 DEG C, at this point, pipe can be observed
Middle mixed liquor 103 is layered, and upper layer is oily phase layer 105, and lower layer is aqueous layer 106.After incubation, liquid relief is used in a heated condition
Rifle takes out lower layer's aqueous phase solution 106, or takes out lower section aqueous phase solution 106 into new centrifuge tube completely after being cooled to room temperature.
If it is desired, 2 μ L RNase A can also be added in aqueous phase solution, 5min is placed at room temperature for remove RNA.
200 μ L isopropanols and 20 μ L silica magnetic beads are added in the centrifuge tube for being placed with aqueous layer, are uniformly mixed, room temperature is anti-
Magnetic suck after 5min is answered to remove supernatant, respectively with 200 μ L washing lotions 1 (1.0M NaCl, 50mM MOPS, 15% isopropanol) and washing lotion 2
After (50mM Tris-HCl, 80% ethyl alcohol) carries out 2 cleanings to magnetic bead, interior solution is managed in exhaustion, air-dries magnetic bead 2min, and 50 μ are added
L eluent enters in centrifuge tube, is incubated at room temperature 2min, takes out supernatant after magnetic suck, supernatant is final nucleic acid product.
Embodiment 2
By 150 μ L water phase reagents (1mM EDTA, 50mM PBS, 4M guanidine hydrochloride, 1%SDS), 20 μ L Proteinase K (20mg/
ML) with 150 μ L paraffin oils be added equipped with FFPE tissue samples 102 1.5mL centrifuge tube 101 in, concussion mixing after it is of short duration from
The heart obtains mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 52 DEG C of heating device 104, is incubated for 3 hours under the conditions of 52 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.If it is desired, can be by centrifuge tube
101 take out, and thermal 104 to be added is warming up to after 80 DEG C and is again put into centrifuge tube 101, are incubated for 1 hour.After incubation, adding
Lower layer's aqueous phase solution 106 is taken out with liquid-transfering gun under heat condition, or takes out lower section aqueous phase solution 106 extremely completely after being cooled to room temperature
In new centrifuge tube.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 3
By 300 μ L water phase reagents (10mM EDTA, 50mM Tris-HCl, 1%SDS), 20 μ L Proteinase Ks (20mg/mL)
It is added in the 1.5mL centrifuge tube 101 equipped with FFPE tissue samples 102 with 150 μ L dimethylbenzene, of short duration centrifugation after concussion mixing obtains
To mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 56 DEG C of heating device 104, is incubated for 2 hours under the conditions of 56 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.If it is desired, can be by centrifuge tube
101 take out, and thermal 104 to be added is warming up to after 90 DEG C and is again put into centrifuge tube 101, are incubated for 30 minutes.After incubation, adding
Lower layer's aqueous phase solution 106 is taken out with liquid-transfering gun under heat condition, or takes out lower section aqueous phase solution 106 extremely completely after being cooled to room temperature
In new centrifuge tube.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 4
By 300 μ L water phase reagents (20mM EDTA, 50mM Tris-HCl, 1%Tween-20,2M guanidine hydrochloride), 20 μ L eggs
White enzyme K (20mg/mL) and 150 μ L silicone oil are added in the 1.5mL centrifuge tube 101 equipped with FFPE tissue samples 102, concussion mixing
Of short duration centrifugation afterwards obtains mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 56 DEG C of heating device 104, is incubated overnight under the conditions of 56 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.After incubation, in heating condition
It is lower to take out lower layer's aqueous phase solution 106 with liquid-transfering gun, or take out lower section aqueous phase solution 106 completely after being cooled to room temperature to new centrifugation
Guan Zhong.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 5
By 200 μ L water phase reagents (20mM EDTA, 50mM Tris-HCl, 2%Tween-20,1M guanidine hydrochloride), 20 μ L eggs
White enzyme K (20mg/mL) and 50 μ L silicone oil are added in the 1.5mL centrifuge tube 101 equipped with FFPE tissue samples 102, after concussion mixing
Of short duration centrifugation obtains mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 56 DEG C of heating device 104, is incubated for 2 hours under the conditions of 56 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.After incubation, in heating condition
It is lower to take out lower layer's aqueous phase solution 106 with liquid-transfering gun, or take out lower section aqueous phase solution 106 completely after being cooled to room temperature to new centrifugation
Guan Zhong.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 7
By 200 μ L water phase reagents (10mM EDTA, 50mM Tris-HCl, 0.5%SDS, 1M guanidinium isothiocyanate), 20 μ L eggs
White enzyme K (20mg/mL) and 50 μ L silicone oil are added in the 1.5mL centrifuge tube 101 equipped with FFPE tissue samples 102, after concussion mixing
Of short duration centrifugation obtains mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 56 DEG C of heating device 104, is incubated for 2 hours under the conditions of 56 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.After incubation, in heating condition
It is lower to take out lower layer's aqueous phase solution 106 with liquid-transfering gun, or take out lower section aqueous phase solution 106 completely after being cooled to room temperature to new centrifugation
Guan Zhong.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 8
By 400 μ L water phase reagents (10mM EDTA, 50mM Tris-HCl, 1%SDS, 2M guanidinium isothiocyanate), 20 μ L albumen
Enzyme K (20mg/mL) and 100 μ L silicone oil are added in the 1.5mL centrifuge tube 101 equipped with FFPE tissue samples 102, after concussion mixing
Of short duration centrifugation obtains mixed liquor 103.
Centrifuge tube is put into and is had warmed up into 56 DEG C of heating device 104, is incubated for 2 hours under the conditions of 56 DEG C, at this point, can
Observe that mixed liquor 103 is layered in pipe, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.After incubation, in heating condition
It is lower to take out lower layer's aqueous phase solution 106 with liquid-transfering gun, or take out lower section aqueous phase solution 106 completely after being cooled to room temperature to new centrifugation
Guan Zhong.
Nucleic acid extraction mode based on paramagnetic particle method is same as Example 1, and details are not described herein again.
Embodiment 9
180 μ L water phase reagents (Buffer ATL, QIAGEN FFPE Nucleic acid purification kits), 20 μ L Proteinase Ks (are taken
From QIAGEN FFPE Nucleic acid purification kits) and 1.5mL centrifugation of the 100 μ L mineral oil addition equipped with FFPE tissue samples 102
In pipe 101, of short duration centrifugation after concussion mixing obtains mixed liquor 103.
It is put into and has warmed up into 65 DEG C of heating device 104, be incubated for 30 minutes under the conditions of 65 DEG C, at this point, can be observed
Mixed liquor 103 is layered in pipe, and upper layer is oily phase layer 105, and lower layer is aqueous layer 106.If it is desired, centrifuge tube 101 can be taken out,
Thermal modules to be added are warming up to after 75 DEG C and are again put into centrifuge tube 101, are incubated for 45 minutes.After incubation, use in a heated condition
Liquid-transfering gun takes out lower layer's aqueous phase solution 106, or takes out lower section aqueous phase solution 106 to new centrifuge tube completely after being cooled to room temperature
In.
Using QIAGEN FFPE Nucleic acid purification kits (QIAGEN；QIAamp DNA FFPE Tissue Kit；
Cat.No.56404) carry out nucleic acid separation, use centrifugal process, with silica gel column method separate nucleic acid, concrete operation method referring to
Operation instruction appended by manufacturer, is summarized as follows:
200 μ L Buffer AL and 200 μ L ethyl alcohol are added in upper step aqueous phase solution, is uniformly mixed, is put into silicagel column,
It is centrifuged (revolving speed 8000rpm) 1 minute, 500 μ L W1 buffers are added in the collecting pipe renewed (collection tube)
500 μ L W2 are added in (Buffer W1), centrifugation (revolving speed 8000rpm) 1 minute, the collecting pipe renewed (collection tube)
Buffer (Buffer W2), centrifugation (revolving speed 8000rpm) 1 minute, the collecting pipe renewed (collection tube) is added without
Reagent, centrifugation (revolving speed 13000rpm) 3 minutes, silicagel column is put into new centrifuge tube, 50 μ L eluent (Elution are added
Buffer it) on the filter membrane of silicagel column, is stored at room temperature 2 minutes.It is centrifuged (revolving speed 13000rpm) 1 minute, liquid is in centrifuge tube
For nucleic acid product.
Embodiment 10
By 210 μ L water phase reagent Protease Solution (applied biosystems；MagMAX FFPE DNA/
RNA Ultra kit；Cat.No.A31881), 20 μ L Proteinase Ks (20mg/mL) and 420 μ L silicone oil are added organizes equipped with FFPE
In the 1.5mL centrifuge tube 101 of sample 102, of short duration centrifugation after concussion mixing obtains mixed liquor 103.
Centrifuge tube 101 is put into and is had warmed up into 56 DEG C of heating device 104, is incubated for 2 hours under the conditions of 56 DEG C, this
When, mixed liquor 103 in pipe can be observed and be layered, upper layer is oily phase layer 105, and lower layer is aqueous layer 106.If it is desired, can will be from
Heart pipe 101 takes out, and thermal 104 to be added is warming up to after 90 DEG C and is again put into centrifuge tube 101, is incubated for 30 minutes.Incubation terminates
Afterwards, take out lower layer's aqueous phase solution 106 with liquid-transfering gun in a heated condition, or after being cooled to room temperature lower section aqueous phase solution 106 is complete
It is complete to take out into new centrifuge tube 101.
Using FFPE Nucleic acid purification kits (the applied biosystems of applied biosystems；MagMAX
FFPE DNA/RNA Ultra kit；Cat.No.A31881 nucleic acid separation) is carried out, paramagnetic particle method is used to separate nucleic acid, it is specific to grasp
Make method referring to operation instruction appended by manufacturer, be summarized as follows:
270 μ L (according to different FFPE size variations) DNA Binding is added in the centrifuge tube that upper step is placed with aqueous layer
Buffer is uniformly mixed, and room temperature mixes reaction 5min, and magnetic suck removes supernatant, (is become respectively according to different FFPE sizes with 400 μ L
Change) DNA Wash Buffer and 500 μ L (according to different FFPE size variations) Wash Solution 2 to magnetic bead carry out 2 times it is clear
After washing, interior solution is managed in exhaustion, air-dries magnetic bead 1-3min, and 50 μ L Elution Solution are added and enter in centrifuge tube, room temperature is mixed
Even incubation 5min takes out supernatant after magnetic suck, and supernatant is final nucleic acid product.
Reference examples 11
Traditional dimethylbenzene process for dewaxing
This reference examples is that gradually process for dewaxing carries out dewaxing treatment to FFPE tissue samples with a kind of traditional dimethylbenzene, subsequent
It is separated with silicagel column method and obtains nucleic acid product, implementation steps are as follows:
1mL dimethylbenzene is added in the 1.5mL centrifuge tube equipped with FFPE tissue samples, after concussion mixing 10 seconds, centrifugation (turns
Fast 14000rpm) 2 minutes, remove supernatant.1mL dehydrated alcohol is added, after concussion mixing, 2 points of centrifugation (revolving speed 14000rpm)
Clock removes supernatant.It is dried at room temperature for sample 30 minutes, 180 μ L water phase reagent (Buffer ATL, QIAGEN FFPE is added
Nucleic acid purification kits), 20 μ L Proteinase Ks, be put into and have warmed up into 56 DEG C of heating module, be incubated under the conditions of 56 DEG C 1 small
When.Centrifuge tube is taken out, centrifuge tube is put by thermal modules to be added again after being warming up to 90 DEG C, is incubated for 1 hour.
It is subsequent to use QIAGEN FFPE Nucleic acid purification kits (QIAGEN；QIAamp DNA FFPE Tissue Kit；
Cat.No.56404 nucleic acid separation) is carried out, specific nucleic acid extraction step is shown as used in the description, and therefore not to repeat here.
Embodiment 12
The present embodiment compares influence of the different FFPE sample process for dewaxing to nucleic acid concentration is extracted.As shown in Fig. 2, this Shen
It please compare single temperature of the invention and be incubated for method and two step silicone oil extraction methods (see specification background technique) and traditional dimethylbenzene
Final nucleic acid concentration after nucleic acid extraction, from Fig. 2 and Fig. 3 it is found that single temperature of the invention is incubated for the nucleic acid that method is finally extracted
Concentration is significantly larger than two step silicone oil lost-wax processes, suitable with the method for traditional dimethylbenzene dewaxing, and extracts gained purity and other two
A method is suitable.The present invention has significant technical advantage compared with traditional technology, not only enormously simplifies experimental implementation process,
Whole dewaxing duration is shortened, final nucleic acid concentration can also be much higher than the prior art.
Although only enumerating manual extraction scheme in the embodiment of the present invention, the present invention is very suitable for and full-automatic FFPE
Dewaxing-nucleic acid-extracting apparatus combines, and realizes full automatic FFPE dewaxing-nucleic acid extraction process.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of fast dewaxing method that FFPE sample nucleic acid extracts, which comprises the following steps:
Step 1: FFPE sample and water phase reagent and oily phase reagent are sufficiently mixed, the first mixed liquor is formed；
Step 2: the first mixed liquor is heated to the arbitrary temp within the scope of 50-95 DEG C, it is sufficiently incubated for 10min- at such a temperature
72h forms the second separating liquid, and second separating liquid upper layer is oily phase, and lower layer is water phase；
Step 3: taking out lower layer's water phase, nucleic acid extraction is carried out；
The oil phase reagent is the oil-phase solution with immiscible, the dissolvable paraffin of water, and the water phase reagent is the cracking of FFPE sample
Liquid and with paraffin is immiscible, water-soluble aqueous phase solution, the water phase reagent includes pH buffer, surfactant and metal
Ion chelating agent.
2. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that the oil phase reagent
For in mineral oil, paraffin oil, vegetable oil and silicone oil any one or it is a variety of.
3. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that the surface-active
Agent is tween, span, Triton, lauryl sodium sulfate, the one or more of neopelex.
4. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that the water phase reagent
It further include protein denaturant.
5. the fast dewaxing method that FFPE sample nucleic acid as claimed in claim 4 extracts, which is characterized in that the albumen qualitative change
Property agent be one or more of rhodanate, isothiocyanate or perchlorate.
6. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that the water phase reagent
It further include Proteinase K.
7. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that the oil phase reagent
With the ratio of water phase reagent in 1:4 between 2:1.
8. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that in the second step
The range of arbitrary temp is 52-65 DEG C.
9. the fast dewaxing method that FFPE sample nucleic acid as described in claim 1 extracts, which is characterized in that in the second step
The time of incubation is 30min-2h.
10. the fast dewaxing kit that a kind of FFPE sample nucleic acid extracts, which is characterized in that the kit uses such as right
It is required that the fast dewaxing method that FFPE sample nucleic acid described in 1-9 any one extracts.
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| CN109957563A (en) * | 2019-05-05 | 2019-07-02 | 上海睿璟生物科技有限公司 | DNA extraction method in paramagnetic particle method FFPE tissue |
| CN110819622A (en) * | 2019-10-09 | 2020-02-21 | 广州达正生物科技有限公司 | Method for rapidly extracting RNA (ribonucleic acid) of paraffin tissue |
| CN111057704A (en) * | 2019-12-31 | 2020-04-24 | 广州市金圻睿生物科技有限责任公司 | A dewaxing lysis binding solution, kit and method for DNA extraction from paraffin section samples |
| CN113088515A (en) * | 2021-05-07 | 2021-07-09 | 杭州康代思锐生物科技有限公司 | Kit and method for extracting FFPE tissue sample DNA and application thereof |
| CN114752466A (en) * | 2022-04-11 | 2022-07-15 | 中国科学院苏州生物医学工程技术研究所 | System for automatically extracting nucleic acid from paraffin-embedded sample |
| CN118006598A (en) * | 2024-02-27 | 2024-05-10 | 国药(武汉)医学实验室有限公司 | Dewaxing solution and dewaxing method for nucleic acid extraction from FFPE samples |
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| CN114752466A (en) * | 2022-04-11 | 2022-07-15 | 中国科学院苏州生物医学工程技术研究所 | System for automatically extracting nucleic acid from paraffin-embedded sample |
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