CN105039315A - Plant bleeding sap RNA extracting method - Google Patents
Plant bleeding sap RNA extracting method Download PDFInfo
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- CN105039315A CN105039315A CN201510589286.6A CN201510589286A CN105039315A CN 105039315 A CN105039315 A CN 105039315A CN 201510589286 A CN201510589286 A CN 201510589286A CN 105039315 A CN105039315 A CN 105039315A
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Abstract
The invention discloses a plant bleeding sap RNA extracting method. The plant bleeding sap RNA extracting method comprises the following steps that freeze frying is conducted on plant bleeding sap, and SDS is added to perform water bathing; water saturated phenol is added and stirred, and then chloroform is added and stirred evenly for centrifugation; supernatant is taken into a novel centrifugation tube and added with water saturated phenol, even stirring is performed, then chloroform is added and stirred evenly for centrifugation; supernatant is taken and added with chloroform, even stirring is performed for centrifugation, and supernatant is taken and added with the same volume of isopropanol for standing; centrifugation is performed, the supernatant is poured off, 80% of ethyl alcohol is adopted for washing and precipitation, 100% of ethyl alcohol is adopted for rewashing, centrifugation and air drying are performed for RNA precipitation, and DEPC water is added. The plant bleeding sap RNA extracting method can inhibit degradation of the RNA in bleeding sap and improve the extracting efficiency of the RNA in bleeding sap. RNA extracting procedures are simplified, the obtained RNA amount is increased, and the amount of RNA extracted from the bleeding sap can meet the inverse transcription demand.
Description
Technical field
The present invention relates to agricultural technology field, be specifically related to a kind of plant bleeding sap RNA extraction method.
Background technology
At present, extract plant tissue RNA method very ripe, but applying these methods extracts RNA in the plant such as grape, cucumber bleeding sap, effect is undesirable, occurs problem: 1) can not carry RNA; 2) in bleeding sap, RNA degraded is fast; 3) the RNA quantity not sufficient proposed, can not meet the needs of reverse transcription.
Summary of the invention
Goal of the invention: the object of this invention is to provide a kind of plant bleeding sap RNA extraction method.
Technical scheme: in order to solve the problems of the technologies described above, the invention provides a kind of plant bleeding sap RNA extraction method, comprises the following steps:
1) by the lyophilize of plant bleeding sap to the 1/8-1/10 of original volume, add the SDS of 1300-1500 μ l, water-bath 65 DEG C of 25 ~ 30min;
2) add the water-saturated phenol of 200-400 μ l, vortex, then add 200-400 μ l chloroform, vortex mixes, 4 DEG C, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
3) get 1100-1300 μ l supernatant liquor in new centrifuge tube, add the water-saturated phenol of 1/3 volume, vortex mixes, then adds the chloroform of supernatant liquor 1/3 volume, and vortex mixes, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
4) get the water-saturated phenol that supernatant 900-1100 μ l adds supernatant liquor 1/2 volume, vortex mixes, then adds the chloroform vortex mixing of supernatant liquor 1/2 volume, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
5) get supernatant 700-900 μ l and add equal-volume chloroform, vortex mixes, and the centrifugal 10min of 12000 ~ 13000rpm, gets supernatant 600-800 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugal 20min of 12000 ~ 13000rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 12000 ~ 13000rpm, 5 ~ 8min, and 100% ethanol is washed once again, the centrifugal 5min of 12000 ~ 13000rpm, dries RNA precipitation, adds the DEPC water of 30 μ l.
Wherein, as preferably, the plant bleeding sap lyophilize in step 1), to 1/10 of original volume, adds the SDS of 1400 μ l, water-bath 65 DEG C of 30min.
Wherein, as preferably, step 2) in add 300 μ l water-saturated phenols, vortex, then add 300ul chloroform, vortex mixes, 4 DEG C, the centrifugal 10min of 13000rpm.
Wherein, as preferably, get 1200 μ l supernatants in new centrifuge tube, add 400 μ l water-saturated phenols in step 3), vortex mixes, then adds 400 μ l chloroforms, and vortex mixes, the centrifugal 10min of 13000rmp.
Wherein, as preferably, step 4) is got supernatant 1000 μ l and is added 500 μ l water-saturated phenols, and vortex mixes, then adds the mixing of 500ul chloroform vortex, the centrifugal 10min of 13000rpm.
Wherein, as preferably, step 5) is got supernatant 800 μ l and is added 800 μ l chloroforms, and vortex mixes, and the centrifugal 10min of 13000rpm, gets supernatant 700 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h.
Wherein, as preferably, the centrifugal 20min of step 6) 13000rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 13000rpm, 5min, and 100% ethanol is washed once again, the centrifugal 5min of 13000rpm, dries RNA precipitation, adds 30 μ lDEPC water.
Beneficial effect: plant bleeding sap RNA extraction method of the present invention can suppress RNA degraded in bleeding sap, improve RNA extraction efficiency in bleeding sap, this invention simplifies RNA extraction procedure, improve RNA and obtain quantity, bleeding sap RNA extracted amount can meet reverse transcription needs.
Accompanying drawing explanation
The RNA detected through gel electrophoresis figure that Fig. 1 embodiment of the present invention 1 ~ 3 is extracted;
After the RNA reverse transcription that Fig. 2 embodiment of the present invention 1 ~ 3 is extracted, cDNA detected through gel electrophoresis figure;
The RNA detected through gel electrophoresis figure that Fig. 3 comparative example is extracted;
After the RNA reverse transcription that Fig. 4 comparative example is extracted, cDNA detected through gel electrophoresis figure.
Embodiment
Below technical solution of the present invention is described in detail, but protection scope of the present invention is not limited to described embodiment.
embodiment 1:
One kind of plant bleeding sap RNA extraction method, comprises the following steps:
1) by the lyophilize of 5ml plant bleeding sap to 1/10 of original volume, add 1400 μ lSDS, water-bath 65 DEG C of 30min;
2) add 300 μ l water-saturated phenols, vortex, then add 300 μ l chloroforms, vortex mixes, 4 DEG C, the centrifugal 10min of 13000rpm;
3) get 1200 μ l supernatants in new centrifuge tube, add 400 μ l water-saturated phenols, vortex mixes, then adds 400 μ l chloroforms, and vortex mixes, the centrifugal 10min of 13000rmp;
4) get supernatant 1000 μ l and add 500 μ l water-saturated phenols, vortex mixes, then adds 500 μ l chloroform vortex mixings, the centrifugal 10min of 13000rpm;
5) get supernatant 800 μ l and add 800 μ l chloroforms, vortex mixes, and the centrifugal 10min of 13000rpm, gets supernatant 700 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugal 20min of 13000rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 13000rpm, 5min, and 100% ethanol is washed once again, the centrifugal 5min of 13000rpm, dries RNA precipitation, adds 30 μ lDEPC water.
Embodiment 2
One kind of plant bleeding sap RNA extraction method, comprises the following steps:
1) by the lyophilize of plant bleeding sap to 1/8 of original volume, add the SDS of 1300 μ l, water-bath 65 DEG C of 25min;
2) add the water-saturated phenol of 200 μ l, vortex, then add 200 μ l chloroforms, vortex mixes, 4 DEG C, the centrifugal 8min of 12000rpm;
3) get 1100 μ l supernatant liquors in new centrifuge tube, add the water-saturated phenol of 1/3 volume, vortex mixes, then adds the chloroform of supernatant liquor 1/3 volume, and vortex mixes, the centrifugal 8min of 12000rpm;
4) get the water-saturated phenol that supernatant 900 μ l adds supernatant liquor 1/2 volume, vortex mixes, then adds the chloroform vortex mixing of supernatant liquor 1/2 volume, the centrifugal 8min of 12000rpm;
5) get supernatant 700 μ l and add equal-volume chloroform, vortex mixes, and the centrifugal 10min of 12000rpm, gets supernatant 600 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugal 20min of 12000rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 12000rpm, 8min, and 100% ethanol is washed once again, the centrifugal 5min of 12000rpm, dries RNA precipitation, adds the DEPC water of 30 μ l.
Embodiment 3
One kind of plant bleeding sap RNA extraction method, comprises the following steps:
1) by the lyophilize of plant bleeding sap to 1/9 of original volume, add the SDS of 1500 μ l, water-bath 65 DEG C of 30min;
2) add the water-saturated phenol of 400 μ l, vortex, then add 400 μ l chloroforms, vortex mixes, 4 DEG C, the centrifugal 9min of 12500rpm;
3) get 1300 μ l supernatant liquors in new centrifuge tube, add the water-saturated phenol of 1/3 volume, vortex mixes, then adds the chloroform of supernatant liquor 1/3 volume, and vortex mixes, the centrifugal 9min of 12500rpm;
4) get the water-saturated phenol that supernatant 1100 μ l adds supernatant liquor 1/2 volume, vortex mixes, then adds the chloroform vortex mixing of supernatant liquor 1/2 volume, the centrifugal 9min of 12500rpm;
5) get supernatant 900 μ l and add equal-volume chloroform, vortex mixes, and the centrifugal 10min of 12500rpm, gets supernatant 800 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugal 20min of 12500rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 12500rpm, 7min, and 100% ethanol is washed once again, the centrifugal 5min of 12500rpm, dries RNA precipitation, adds the DEPC water of 30 μ l.
The present embodiment obtains the amount of RNA between 1.8-2.2 μ g/ μ l, obtains the success ratio that can meet reverse transcription RNA concentration requirement and reaches more than 99%, RNA degradation rate and be less than 0.5%.
Comparative example:
1. water-bath is adjusted to 65 DEG C;
2. in 2ml centrifuge tube, add 1100 μ l lysate SDS, then add 50 μ l beta-mercaptoethanol mixings, or in 2ml centrifuge tube, add 1100 μ l lysate CTAB, then add 30 μ l beta-mercaptoethanol CTAB;
3. grind away, is added to centrifuge tube, and timing puts 30 ~ 45min in water-bath, its interval 10min transpose;
4. add chloroform about 850 μ l after taking out cooling, vortex mixes, 12000r/m, centrifugal 20min;
5. in new 2ml centrifuge tube, add 450 μ l phenolic acid (water-saturated phenol, lower floor is phenol), with supernatant liquor (1000 μ l) vortex, 12000r/m, centrifugal (18min ~ 20min);
6. in new 2ml centrifuge tube, add 900 μ l chloroforms, add supernatant liquid (about 850 μ l) vortex, 12000r/m, 18min(15 ~ 20min);
7. in new 1.5ml centrifuge tube, add 35 μ lNaAC and 70 μ l dehydrated alcohols, get supernatant liquid about 700 μ l and add wherein, put upside down mixing, place on water box ,-20 DEG C of standing more than 60min();
The centrifugal 20min of 8.12000r/m, gets supernatant liquid about 700 μ l and adds equal-volume Virahol and put upside down mixing ,-20 DEG C, crosses liquid precipitate (more than 5h);
The centrifugal 20min of 12000r/m, outwells supernatant liquid, adds 750 μ l dehydrated alcohol+250 μ lDEPC water to precipitation, flick → 12000r/m, 6min abandons supernatant, is placed in 750 μ l dehydrated alcohols that paper slightly absorbs water → add, 12000r/m, 6min → abandon supernatant, on paper dry (about more than 10min), add 45 μ lDEPC water, dissolution precipitation, put into 65 DEG C of water-bath 10min, then-20 DEG C of preservations.
The embodiment of the present invention 1 ~ 3 extracts bleeding sap RNA see Fig. 1: RNA detected through gel electrophoresis of carrying (A, B, C repeat for tri-times to extract) gel electrophoresis images band is clear, and brightness is high, and RNA extracted amount foot is described; Fig. 2: extract RNA reverse transcription after, cDNA detected through gel electrophoresis: cDNA band is clear, illustrates that reverse transcription is effective.
Comparative example extracts bleeding sap RNA see Fig. 3: bleeding sap RNA(A, B, C repeat for tri-times to extract) gel electrophoresis images band is fuzzy, low lightness, and RNA extracted amount deficiency is described; After Fig. 4 extracts RNA reverse transcription, cDNA detected through gel electrophoresis: have no cDNA band, illustrates that the amount of RNA extraction from bleeding sap does not reach the requirement of reverse transcription.
Claims (7)
1. a kind of plant bleeding sap RNA extraction method, is characterized in that: comprise the following steps:
1) by the lyophilize of plant bleeding sap to the 1/8-1/10 of original volume, add the SDS of 1300-1500 μ l, water-bath 65 DEG C of 25 ~ 30min;
2) add the water-saturated phenol of 200-400 μ l, vortex, then add 200-400 μ l chloroform, vortex mixes, 4 DEG C, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
3) get 1100-1300 μ l supernatant liquor in new centrifuge tube, add the water-saturated phenol of 1/3 volume, vortex mixes, then adds the chloroform of supernatant liquor 1/3 volume, and vortex mixes, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
4) get the water-saturated phenol that supernatant 900-1100 μ l adds supernatant liquor 1/2 volume, vortex mixes, then adds the chloroform vortex mixing of supernatant liquor 1/2 volume, the centrifugal 8 ~ 10min of 12000 ~ 13000rpm;
5) get supernatant 700-900 μ l and add equal-volume chloroform, vortex mixes, and the centrifugal 10min of 12000 ~ 13000rpm, gets supernatant 600-800 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugal 20min of 12000 ~ 13000rpm, outwells supernatant, and 80% ethanol 1ml washes precipitation, 12000 ~ 13000rpm, 5 ~ 8min, and 100% ethanol is washed once again, the centrifugal 5min of 12000 ~ 13000rpm, dries RNA precipitation, adds the DEPC water of 30 μ l.
2. a kind of plant bleeding sap RNA extraction method according to claim 1, is characterized in that: the plant bleeding sap lyophilize in described step 1), to 1/10 of original volume, adds the SDS of 1400 μ l, water-bath 65 DEG C of 30min.
3. a kind of plant bleeding sap RNA extraction method according to claim 1, is characterized in that: described step 2) in add 300 μ l water-saturated phenols, vortex, then add 300ul chloroform, vortex mixes, 4 DEG C, the centrifugal 10min of 13000rpm.
4. a kind of plant bleeding sap RNA extraction method according to claim 1, is characterized in that: get 1200 μ l supernatants in described step 3) in new centrifuge tube, add 400 μ l water-saturated phenols, vortex mixes, add 400 μ l chloroforms again, vortex mixes, the centrifugal 10min of 13000rmp.
5. a kind of plant bleeding sap RNA extraction method according to claim 1, is characterized in that: described step 4) is got supernatant 1000 μ l and added 500 μ l water-saturated phenols, and vortex mixes, then adds the mixing of 500ul chloroform vortex, the centrifugal 10min of 13000rpm.
6. a kind of plant bleeding sap RNA extraction method according to claim 1, it is characterized in that: described step 5) is got supernatant 800 μ l and added 800 μ l chloroforms, vortex mixes, the centrifugal 10min of 13000rpm, get supernatant 700 μ l and add equal-volume Virahol ,-20 DEG C of standing more than 5h.
7. a kind of plant bleeding sap RNA extraction method according to claim 1, it is characterized in that: the centrifugal 20min of described step 6) 13000rpm, outwell supernatant, 80% ethanol 1ml washes precipitation, 13000rpm, 5min, 100% ethanol is washed once again, the centrifugal 5min of 13000rpm, dries RNA precipitation, adds 30 μ lDEPC water.
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| CN201510589286.6A CN105039315A (en) | 2015-09-16 | 2015-09-16 | Plant bleeding sap RNA extracting method |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102283408A (en) * | 2011-06-17 | 2011-12-21 | 合肥红峥生物科技有限公司 | Loofah bleeding sap functional beverage and preparation method thereof |
| CN102628039A (en) * | 2012-04-11 | 2012-08-08 | 中国林业科学研究院林业研究所 | General plant total RNA (Ribose Nucleic Acid) extraction method |
| CN102884191A (en) * | 2010-02-26 | 2013-01-16 | 凯杰有限公司 | Process for parallel isolation and/or purification of RNA and DNA |
| CN102871196A (en) * | 2011-07-15 | 2013-01-16 | 伊力哈木·托合迪 | Vitis vinifera bleeding sap drink containing zinc metallothionein (Zn-MT) and preparation method thereof |
| CN103083216A (en) * | 2011-11-01 | 2013-05-08 | 杨秀梅 | All-natural cucumber vine bleeding sap beautifying water |
| CN103911369A (en) * | 2014-04-04 | 2014-07-09 | 中国农业科学院烟草研究所 | Method of effectively extracting total RNA (Ribonucleic Acid) of tobacco mature leaf |
| CN104313014A (en) * | 2014-10-08 | 2015-01-28 | 福建农林大学 | Method for rapidly extracting total ribonucleic acid (RNA) of fir |
| CN104357439A (en) * | 2014-11-27 | 2015-02-18 | 广东省农业科学院作物研究所 | Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols |
-
2015
- 2015-09-16 CN CN201510589286.6A patent/CN105039315A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102884191A (en) * | 2010-02-26 | 2013-01-16 | 凯杰有限公司 | Process for parallel isolation and/or purification of RNA and DNA |
| CN102283408A (en) * | 2011-06-17 | 2011-12-21 | 合肥红峥生物科技有限公司 | Loofah bleeding sap functional beverage and preparation method thereof |
| CN102871196A (en) * | 2011-07-15 | 2013-01-16 | 伊力哈木·托合迪 | Vitis vinifera bleeding sap drink containing zinc metallothionein (Zn-MT) and preparation method thereof |
| CN103083216A (en) * | 2011-11-01 | 2013-05-08 | 杨秀梅 | All-natural cucumber vine bleeding sap beautifying water |
| CN102628039A (en) * | 2012-04-11 | 2012-08-08 | 中国林业科学研究院林业研究所 | General plant total RNA (Ribose Nucleic Acid) extraction method |
| CN103911369A (en) * | 2014-04-04 | 2014-07-09 | 中国农业科学院烟草研究所 | Method of effectively extracting total RNA (Ribonucleic Acid) of tobacco mature leaf |
| CN104313014A (en) * | 2014-10-08 | 2015-01-28 | 福建农林大学 | Method for rapidly extracting total ribonucleic acid (RNA) of fir |
| CN104357439A (en) * | 2014-11-27 | 2015-02-18 | 广东省农业科学院作物研究所 | Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols |
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Application publication date: 20151111 |