CN110317804A - The method and its kit of RNA are extracted from paraffin-embedded tissue - Google Patents
The method and its kit of RNA are extracted from paraffin-embedded tissue Download PDFInfo
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- CN110317804A CN110317804A CN201910579510.1A CN201910579510A CN110317804A CN 110317804 A CN110317804 A CN 110317804A CN 201910579510 A CN201910579510 A CN 201910579510A CN 110317804 A CN110317804 A CN 110317804A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The method that the invention discloses a kind of to extract RNA from paraffin-embedded tissue, including paraffin-embedded tissue is cut to obtain with a thickness of 3-10 μm of paraffin section sample, and the paraffin section sample is put into microcentrifugal tube；Be added dimethylbenzene in the microcentrifugal tube, 55 DEG C heating water bath 3 minutes to dissolve the paraffin in the paraffin section sample, centrifugation removes supernatant after forming tissue precipitating to remove paraffin；Dehydrated alcohol is added into tissue precipitating and is vortexed, then removes supernatant after being centrifuged；It air-dries at room temperature and obtains sample.The higher RNA of quality can be extracted from paraffin-embedded tissue using such mode, the RNA concentration extracted is higher, and purity is better.
Description
Technical field
The present invention relates to field of biotechnology more particularly to it is a kind of from paraffin-embedded tissue extract RNA method and
Its kit.
Background technique
Be born from the DNA double spin model of Watson, Crick, biology has entered a completely new epoch, to DNA and
The basis of RNA being extracted into as all subject scientific researches in the fields such as biomedicine field even farming, forestry, husbandary and fishing.In the past 10 years,
With the fast development of gene diagnosis, GM food detection, personalized medicine etc., modern molecular biology technique is more and more wider
It is used for all fields of human diseases research generally, has accumulated new data to understand the variation of genome under pathological state.Fu Er
Malin fixed paraffin-embedded tissue (formalinfixedandparaffinembeddedtissues, FFPE) is medical institutions
The most common method for saving pathological tissue.The a large amount of paraffin-embedded tissues accumulated in hospital pathology department archives are one reliable
Molecular biology research material source.Since fresh specimens are difficult to obtain, to carry out entity molecule biological study, to
When having case to carry out retrospective study, gene extraction can only be carried out from paraffin-embedded tissue.In addition, for clinical and scientific research and
Speech, the paraffin-embedded tissue holding time is long, and Conservation environment is of less demanding, very convenient operation, therefore paraffin-embedded tissue just at
For the most abundant precious resources in source.The nucleic acid that high purity and high quality is isolated from the sample of paraffin-embedded tissue is downstream examination
Test the most basic premise gone on smoothly.Due to the single-stranded structure of RNA is unstable and nature in there are a large amount of RNA enzyme, make
The extraction difficulty of RNA is greater than DNA, and most of product is still served only for the extraction of DNA, for the relatively seldom of RNA extraction, from paraffin
The product that RNA is extracted in investing tissue is still seldom.
Summary of the invention
In order to overcome the defects of the prior art, the embodiment of the invention provides one kind extracts from paraffin-embedded tissue
The method of RNA, comprising the following steps:
The paraffin-embedded tissue is cut to obtain the paraffin section sample with a thickness of 3~10 μm, and the paraffin is cut
Piece sample is put into microcentrifugal tube；
Dimethylbenzene is added in the microcentrifugal tube for being equipped with the paraffin section sample；
55 DEG C of paraffin section sample heating water bath 3 minutes is cut with dissolving the paraffin after the dimethylbenzene is added
Paraffin in piece sample；
The paraffin section sample for being dissolved in the dimethylbenzene is centrifuged, layering removes after forming tissue precipitating
Clear liquid, to remove paraffin；
It is added after dehydrated alcohol and is vortexed to tissue precipitating in opaque state into tissue precipitating, it is right
Tissue precipitating in opaque state is centrifuged, and supernatant is removed after layering, the dehydrated alcohol is for washing stone
Wax is sliced sample；
It air-dries at room temperature, the dehydrated alcohol that volatilizees obtains sample.
Further, step " paraffin-embedded tissue is cut to obtain the paraffin section sample with a thickness of 3~10 μm ",
The paraffin section sample with a thickness of 5~6 μm.
Further, step " dimethylbenzene is added in the microcentrifugal tube for being equipped with the paraffin section sample ", adds
Enter 1mL dimethylbenzene；Step " carries out heating to the paraffin section sample until the paraffin section after the dimethylbenzene is added
Paraffin dissolution in sample ", under 55 DEG C of water bath conditions, makes the paraffin in the paraffin section sample be dissolved in the dimethylbenzene,
Heating dissolves the paraffin in 3 minutes；Step " is centrifuged the paraffin section sample for being dissolved in the dimethylbenzene, is layered
Supernatant is removed after forming tissue precipitating ", centrifugal speed is 14000~16000rpm, and centrifugation time is 2 minutes.
Further, step " is vortexed after dehydrated alcohol is added into tissue precipitating to the tissue place of settling
In opaque state, the tissue precipitating in opaque state is centrifuged, removes supernatant after separating the layers ", it is added
The dehydrated alcohol of 1mL, centrifugal speed are 14000~16000rpm, and centrifugation time is 2 minutes.
Further, RNA extraction is carried out to the sample obtained through above-mentioned steps.
Further, lysate is added in the centrifuge tube without RNA enzyme equipped with the sample, and whirlpool mixes, heating
Supernatant is taken to be transferred in the centrifuge tube of new no RNA enzyme after centrifugation；
Magnetic bead and Hybridization Buffer mixed liquor are added in the centrifuge tube without RNA enzyme equipped with the supernatant, at room temperature
It is mixed to get RNA magnetic bead combination, the magnetic bead and the Hybridization Buffer mixed liquor are 1:1；
The RNA magnetic bead combination is washed with washing buffer, and removes supernatant；
The RNA magnetic bead combination after washing is eluted with eluent, the RNA magnetic bead that suspends again combines
Body collects supernatant after heating to it, obtain the RNA in paraffin-embedded tissue.
Further, the lysate uses Trizol reagent, adds in the centrifuge tube without RNA enzyme equipped with the sample
The Trizol reagent for entering 1mL, blows and beats repeatedly, and 200 μ L chloroformic solutions are added after 15 minutes in 4 DEG C of standings, acutely shakes room temperature after 15s
It places 5 minutes, 4 DEG C are extremely layered for centrifugation 15 minutes, and supernatant is taken to be transferred in the centrifuge tube of new no RNA enzyme.
Further, the Hybridization Buffer mixed liquor is diluted to 0.5* Hybridization Buffer mixed liquor by 20*SSC.
Further, the washing buffer uses the dehydrated alcohol of 100 μ L, and separates removal supernatant with magnetic frame,
And repeat this step twice.
Further, the eluent using the RNase free of 20~50 μ L DEPC treated aqua sterilisa, and
Supernatant is collected using magnetic frame rapidly after heating 2 minutes at 65 DEG C, and supernatant is transferred to the centrifugation of new no RNA enzyme
Guan Zhong obtains the RNA in paraffin-embedded tissue.
The invention also includes a kind of kits for extracting RNA, including the lysate, the magnetic bead, the Hybridization Buffer
Mixed liquor, the washing buffer and the eluent；
The lysate includes A treatment fluid and B treatment fluid, and the A treatment fluid is Trizol reagent, and the B treatment fluid is
Chloroformic solution；
The magnetic bead is 1 μm of the silicone hydroxyl magnetic bead of 30mg/mL, and the Hybridization Buffer mixed liquor is to be diluted to by 20*SSC
0.5* Hybridization Buffer mixed liquor, the formula of the 20*SSC are as follows: taking 3M sodium chloride 175g/L, 0.3M sodium citrate 88g/L, use
Then plus hydrochloric acid tune PH to 7.0-7.4 500ml distilled water after mixing, is settled to 1l,；The magnetic bead and the Hybridization Buffer
Mixed liquor is mixed to form in conjunction with liquid；
The dehydrated alcohol that the washing buffer is 100%；
DEPC treated the aqua sterilisa that the eluent is RNase free.
Beneficial effects of the present invention are as follows: paraffin-embedded tissue is from a wealth of sources, and convenient for saving, can be from paraffin embedding
RNA is extracted in tissue, is solved and is extracted defect existing for RNA from fresh cells, it is few mainly due to fresh cells source, and
Not easy to maintain, it is impossible to meet the needs of current research.And the method behaviour of the invention that RNA is extracted from paraffin-embedded tissue
Work is simple, the used time is short, and extraction effect is good.
For above and other objects, features and advantages of the invention can be clearer and more comprehensible, preferred embodiment is cited below particularly,
And cooperate institute's accompanying drawings, it is described in detail below.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the flow chart that paraffin tissue sections are handled in the embodiment of the present invention；
Fig. 2 is that A group and the resulting RNA product of B group carry out electrophoresis detection comparative result figure in the embodiment of the present invention；
Fig. 3 is that the resulting RNA product of S group carries out electrophoresis detection result figure in the embodiment of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that the side of the instructions such as term " on ", "lower", "bottom", "inner", "outside"
Position or positional relationship are to be based on the orientation or positional relationship shown in the drawings, and are merely for convenience of description of the present invention and simplification of the description,
Rather than the device or element of indication or suggestion meaning must have a particular orientation, be constructed and operated in a specific orientation, because
This is not considered as limiting the invention.In addition, term " first ", " second " etc. are used for description purposes only, and cannot understand
For indication or suggestion relative importance or implicitly indicate the quantity of indicated technical characteristic.Define as a result, " first ",
One or more of the features can be expressed or be implicitly included to the feature of " second " etc..
In order to achieve the above objectives, the present invention provides a kind of extracts the kit of RNA and its is mentioned from paraffin-embedded tissue
The method for taking RNA.
The embodiment of the present invention also provides a kind of method that RNA is extracted from paraffin-embedded tissue, including extracts pre-treatment stone
The method that wax investing tissue obtains the method for paraffin tissue sections sample and extracts RNA.
The method for extracting pre-treatment paraffin-embedded tissue specifically comprises the following steps:
It cuts the paraffin-embedded tissue using slicer to obtain with a thickness of 3~10 μm of paraffin section samples, and will be described
Paraffin section sample is put into 1.5mL microcentrifugal tube；As preferred embodiment a kind of in the present embodiment, it is proposed that use 5
~6 μm of paraffin section samples.
In the microcentrifugal tube for being equipped with the paraffin section sample be added 1mL dimethylbenzene, and briefly be vortexed, from
The heart is to ensure all paraffin section sample submergences in a solvent.
The paraffin section sample of above-mentioned addition dimethylbenzene is subjected to 55 DEG C of heating water baths, is heated 3 minutes, if paraffin organization is not
Dissolution, can continue water-bath three minutes, until the paraffin dissolution in the paraffin section sample.
The paraffin section sample for being dissolved in the dimethylbenzene is centrifuged with maximum speed, it is preferred that with 14000
~16000rpm is centrifuged 2 minutes, and layering forms tissue precipitating.Later tissue precipitating should form dense granule for centrifugation, if do not had
Dense granule is formed, then needs to be centrifuged again 2 minutes.Supernatant is removed with liquid-transfering gun after centrifugation, this step is for removing
Paraffin.
100% dehydrated alcohol of 1mL is added into tissue precipitating and is vortexed and is in opaque state to tissue precipitating.
The tissue in opaque state is precipitated and is centrifuged with maximum speed, it is preferred that with 14000~16000rpm centrifugation
2 minutes.Then in the case where not interference particle, supernatant is removed with liquid-transfering gun as much as possible.Repeat above-mentioned steps into
Second of ethanol washing of row, to ensure to completely remove solvent, this step washs paraffin section sample with dehydrated alcohol.
Guarantee to air-dry 15-25 minutes in the case where sterile no RNA enzyme at room temperature, vapors away dehydrated alcohol and obtain sample.
Effect through the processed obtained paraffin tissue sections sample extraction RNA of the above method is good, and concentration is high, and purity is good
It is good.
The embodiment of the present invention further includes the method using paraffin tissue sections sample extraction RNA obtained by the above method,
Specifically comprise the following steps:
The Trizol reagent of 1mL is added in the centrifuge tube without RNA enzyme equipped with the sample, blows and beats repeatedly, 4 DEG C of standings
200 μ L chloroformic solutions are added after 15 minutes, are placed at room temperature for 5 minutes after acutely shaking 15s, 4 DEG C are extremely layered for centrifugation 15 minutes, take
Clear liquid is transferred in the centrifuge tube of new no RNA enzyme.
This process causes eucaryotic cell structure broken, nucleoprotein is due to its secondary structure for the protein in sample dissolution
It destroys and disappears and separated with nucleic acid rapidly.
Magnetic bead and Hybridization Buffer mixed liquor are added in the supernatant, is mixed to get RNA magnetic bead combination at room temperature,
The magnetic bead and the Hybridization Buffer mixed liquor are 1:1.Preferably, the Hybridization Buffer mixing of the magnetic bead and 20 μ L of 20 μ L is added
Liquid, the magnetic bead and the Hybridization Buffer mixed liquor can increase in right amount according to the size of paraffin tissue sections sample, but at most
No more than 40 μ L.RNA magnetic bead combination is collected with magnetic frame again.
RNA magnetic bead combination is washed with washing buffer, and in present embodiment, the washing buffer is using 100 μ L's
Dehydrated alcohol.Removal supernatant is separated with magnetic frame after washing.
The RNA magnetic bead combination after washing is eluted with eluent, in present embodiment, the eluent is adopted
With the DEPC of the RNase free of 20~50 μ L treated aqua sterilisa, suspend the RNA magnetic bead combination again, and 65
Supernatant is collected after heating 2 minutes at DEG C rapidly, and supernatant is transferred in the centrifuge tube of new no RNA enzyme, obtains paraffin
RNA in investing tissue.
RNA obtained can be saved one month at -20 DEG C, in -80 DEG C of long-term preservations.
The embodiment of the present invention provides a kind of kit that RNA is extracted from paraffin-embedded tissue.The kit includes above-mentioned
Lysate, Hybridization Buffer mixed liquor, washing buffer and eluent in step.
The lysate includes A treatment fluid and B treatment fluid, and the A treatment fluid is Trizol reagent, and the B treatment fluid is
Chloroformic solution；
The magnetic bead is 1 μm of the silicone hydroxyl magnetic bead of 30mg/mL, and the Hybridization Buffer mixed liquor is to be diluted to by 20*SSC
0.5* Hybridization Buffer mixed liquor, the formula of the 20*SSC are as follows: taking 3M sodium chloride 175g/L, 0.3M sodium citrate 88g/L, use
Then plus hydrochloric acid tune PH to 7.0-7.4 500ml distilled water after mixing, is settled to 1L,；The magnetic bead and the Hybridization Buffer
Mixed liquor is mixed to form in conjunction with liquid；
The dehydrated alcohol that the washing buffer is 100%；
DEPC treated the aqua sterilisa that the eluent is RNase free.
First comparative example
The effect of the kit of RNA is extracted in the verifying present invention.
Experimental method
Serial section 12 are carried out to paraffin-embedded tissue, is divided into tri- groups of A, B, C according to every group 4.Wherein, A group uses
Kit in the present embodiment and RNA extraction is carried out using method of the invention, B group and component C Yong not match winged and Tiangeng of writing from memory
Kit carries out RNA extraction.
It should be noted that the kit of Sai Mofei Science and Technology Ltd. extracts and Tiangeng biochemical technology (Beijing) limited public affairs
The kit of department, which extracts, carries out nucleic acid extraction in strict accordance with the specification of kit.
Experimental result
Electrophoresis detection comparative result figure
Kit and Sai Mo to the present embodiment fly the resulting RNA product of kit and carry out electrophoresis detection, and electrophoresis result is asked
Referring to Fig. 2.Wherein c indicate the present embodiment as a result, s indicate match it is silent fly as a result, o indicates oligo (dt) primer, r indicate with
Power traction object.
The RNA sample of tri- groups of extractions of A, B, C is extracted to the survey of RNA concentration and purity through nucleic acid trace test instrument
Fixed, the concentration and purity of measurement are as shown in table 1.
Table 1
As shown in Table 1, (it is greater than using the concentration height of kit provided in this embodiment and the RNA for extracting the acquisition of RNA method
100ng/ μ L), purity is good (2.0~2.2).And the RNA effect extracted significantly larger than matches silent winged kit and Tiangeng examination
Agent box.Illustrate to be suitable for high-throughput extraction RNA in the present embodiment.It is as can be seen from Figure 2, provided in this embodiment in conjunction with electrophoretogram 2
For the product that the obtained sample RNA product of kit winged kit silent compared with match extracts, collimation is good, and brightness is high, shows to extract
RNA integrality it is good, concentration is higher, and without other RNA pollute.Therefore, the RNA of the extraction tissue sample of kit of the present invention
Efficiency is higher than existing kit.
To the kit and extracting method extraction RNA reverse transcription of the invention for using the present embodiment as a result, such as 2 institute of table
Show.
| Sample number | Concentration (ng/ μ L) | Purity |
| A1 | 2474.3 | 1.895 |
| A2 | 2365.7 | 1.871 |
| A3 | 2400.8 | 1.874 |
| A4 | 2227.0 | 1.812 |
Table 2
As shown in Table 2, extract RNA reverse transcription as a result, its concentration has been more than 2000 (ng/ μ L), purity is on 1.8 left sides
It is right.It is met the requirements using the RNA that the kit and method of the invention of the present embodiment extract.
Second comparative example
The method of paraffin-embedded tissue is handled in the verifying present invention before extraction to extraction RNA effect.
Experimental method
Paraffin-embedded tissue is taken, obtains paraffin tissue sections sample according to the method for handling paraffin-embedded tissue in the present invention
S group extracts nucleic acid using paramagnetic particle method provided by the invention.
Experimental result
RNA product resulting to S group carries out electrophoresis detection, and electrophoresis result is referring to figure 3..
From the figure 3, it may be seen that the resulting product of experimental group S group, collimation is good, and brightness is high.Show that the RNA integrality extracted is good,
Concentration is higher, and pollutes without other RNA.
The measurement that the sample of nucleic acid of S group extraction is extracted to nucleic acid concentration and purity through nucleic acid trace test instrument, is surveyed
Fixed concentration and purity is as shown in table 2.
Table 2
As shown in Table 2, extract what RNA was obtained using the method for the present embodiment processing paraffin-embedded tissue and with paramagnetic particle method
The concentration of RNA is high (being greater than 100ng/ μ L), and purity is good (2.0~2.2).RNA reverse transcription as a result, its concentration is more than
2000 (ng/ μ L), purity 1.8 or so, meet standard required for experiment.
To sum up, the method that paraffin-embedded tissue is handled using kit provided in an embodiment of the present invention and before extraction, is mentioned
The RNA concentration got is high, and purity is good, meets the requirements, and the RNA electrophoresis result extracted is also more preferable.
Specific embodiment is applied in the present invention, and principle and implementation of the present invention are described, above embodiments
Explanation be merely used to help understand method and its core concept of the invention；At the same time, for those skilled in the art,
According to the thought of the present invention, there will be changes in the specific implementation manner and application range, in conclusion in this specification
Appearance should not be construed as limiting the invention.
Claims (11)
1. a kind of method for extracting RNA from paraffin-embedded tissue, which comprises the following steps:
The paraffin-embedded tissue is cut to obtain the paraffin section sample with a thickness of 3~10 μm, and by the paraffin section sample
Originally it is put into microcentrifugal tube；
Dimethylbenzene is added in the microcentrifugal tube for being equipped with the paraffin section sample；
To 55 DEG C of paraffin section sample heating water bath 3 minutes to dissolve the paraffin section sample after the dimethylbenzene is added
Paraffin in this；
The paraffin section sample for being dissolved in the dimethylbenzene is centrifuged, layering removes supernatant after forming tissue precipitating
Liquid, to remove paraffin；
It is added after dehydrated alcohol to be vortexed into tissue precipitating and is in opaque state to tissue precipitating, to being in
The tissue precipitating of opaque state is centrifuged, and supernatant is removed after layering, and the dehydrated alcohol is cut for washing paraffin
Piece sample；
It air-dries at room temperature, the dehydrated alcohol that volatilizees obtains sample.
2. the method according to claim 1 for extracting RNA from paraffin-embedded tissue, which is characterized in that step " will be described
Paraffin-embedded tissue cuts to obtain the paraffin section sample with a thickness of 3~10 μm ", the paraffin section sample with a thickness of 5~6
μm。
3. the method according to claim 1 for extracting RNA from paraffin-embedded tissue, which is characterized in that step " is being equipped with
Dimethylbenzene is added in the microcentrifugal tube of the paraffin section sample ", 1mL dimethylbenzene is added；Step " is being added described two
Heating is carried out to the paraffin section sample after toluene until the paraffin in the paraffin section sample dissolves ", in 55 DEG C of water-baths
Under the conditions of, so that the paraffin in the paraffin section sample is dissolved in the dimethylbenzene, heating dissolves the paraffin in 3 minutes；Step is " right
The paraffin section sample for being dissolved in the dimethylbenzene is centrifuged, and layering removes supernatant after forming tissue precipitating ", centrifugation
Speed is 14000~16000rpm, and centrifugation time is 2 minutes.
4. the method according to claim 1 for extracting RNA from paraffin-embedded tissue, which is characterized in that step " Xiang Suoshu
It organizes to be vortexed to tissue precipitating after dehydrated alcohol is added in precipitating in opaque state, in opaque state
The tissue precipitating be centrifuged, remove supernatant after separating the layers ", the dehydrated alcohol of 1mL, centrifugal speed 14000 is added
~16000rpm, centrifugation time are 2 minutes.
5. the method according to any one of claims 1 to 4 for extracting RNA from paraffin-embedded tissue, which is characterized in that
RNA extraction is carried out to the sample obtained through Claims 1-4 any one.
6. the method according to claim 5 for extracting RNA from paraffin-embedded tissue, which is characterized in that including following step
It is rapid:
Lysate is added in the centrifuge tube without RNA enzyme equipped with the sample, heats and whirlpool mixes, take supernatant after centrifugation
It is transferred in the centrifuge tube of new no RNA enzyme；
Magnetic bead and Hybridization Buffer mixed liquor are added in the centrifuge tube without RNA enzyme equipped with the supernatant, mixes at room temperature
RNA magnetic bead combination is obtained, the magnetic bead and the Hybridization Buffer mixed liquor are 1:1；
The RNA magnetic bead combination is washed with washing buffer, and removes supernatant；
The RNA magnetic bead combination after washing is eluted with eluent, suspend the RNA magnetic bead combination again, right
It collects supernatant after heating, obtain the RNA in paraffin-embedded tissue.
7. the method according to claim 6 for extracting RNA from paraffin-embedded tissue, which is characterized in that the lysate
Using Trizol reagent, the Trizol reagent of 1mL is added in the centrifuge tube without RNA enzyme equipped with the sample, blows and beats repeatedly,
200 μ L chloroformic solutions are added after 15 minutes in 4 DEG C of standings, are placed at room temperature for 5 minutes after acutely shaking 15s, and 4 DEG C are extremely divided for centrifugation 15 minutes
Layer, takes supernatant to be transferred in the centrifuge tube of new no RNA enzyme.
8. the method according to claim 6 for extracting RNA from paraffin-embedded tissue, which is characterized in that the hybridization is slow
It rushes mixed liquor and 0.5* Hybridization Buffer mixed liquor is diluted to by 20*SSC.
9. the method according to claim 6 for extracting RNA from paraffin-embedded tissue, which is characterized in that the washing is slow
Fliud flushing uses the dehydrated alcohol of 100 μ L, and separates removal supernatant with magnetic frame, and repeat this step twice.
10. the method according to claim 6 for extracting RNA from paraffin-embedded tissue, which is characterized in that the eluent
Using DEPC treated the aqua sterilisa of the RNase free of 20~50 μ L, and used rapidly after being heated 2 minutes at 65 DEG C
Magnetic frame collects supernatant, and supernatant is transferred in the centrifuge tube of new no RNA enzyme, obtains in paraffin-embedded tissue
RNA。
11. a kind of kit for extracting RNA, which is characterized in that the cracking including such as claim 6~10 any one
Liquid, the magnetic bead, the Hybridization Buffer mixed liquor, the washing buffer and the eluent；
The lysate includes A treatment fluid and B treatment fluid, and the A treatment fluid is Trizol reagent, and the B treatment fluid is chloroform
Solution；
The magnetic bead is 1 μm of the silicone hydroxyl magnetic bead of 30mg/mL, and the Hybridization Buffer mixed liquor is to be diluted to 0.5* by 20*SSC
The formula of Hybridization Buffer mixed liquor, the 20*SSC is as follows: taking 3M sodium chloride 175g/L, 0.3M sodium citrate 88g/L, uses
Then plus hydrochloric acid tune PH to 7.0-7.4 500ml distilled water after mixing, is settled to 1l,；The magnetic bead and the Hybridization Buffer
Mixed liquor is mixed to form in conjunction with liquid；
The dehydrated alcohol that the washing buffer is 100%；
DEPC treated the aqua sterilisa that the eluent is RNase free.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110835628A (en) * | 2019-11-25 | 2020-02-25 | 宁波艾捷康宁生物科技有限公司 | Paraffin removal lysate for extracting genome DNA of paraffin section, extraction kit and extraction method |
| CN113832146A (en) * | 2021-11-15 | 2021-12-24 | 复旦大学附属中山医院 | Method for extracting pathogenic microorganism nucleic acid from formalin-fixed paraffin-embedded sample |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110835628A (en) * | 2019-11-25 | 2020-02-25 | 宁波艾捷康宁生物科技有限公司 | Paraffin removal lysate for extracting genome DNA of paraffin section, extraction kit and extraction method |
| CN113832146A (en) * | 2021-11-15 | 2021-12-24 | 复旦大学附属中山医院 | Method for extracting pathogenic microorganism nucleic acid from formalin-fixed paraffin-embedded sample |
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