CN110819622A - Method for rapidly extracting RNA (ribonucleic acid) of paraffin tissue - Google Patents

Abstract

The invention discloses a method for rapidly extracting RNA of paraffin tissue. A. Dewaxing: putting the FFPE sample into a dewaxing mixed liquor buffer for dewaxing; B. lysis of the tissue: cracking the dewaxed sample by using a cracking solution buffer RLT to obtain a cracking solution; C. and (3) purification: adding the purified solution 1 into the lysate, uniformly mixing and incubating, centrifuging to obtain a supernatant, transferring the supernatant into a new tube, adding chloroform, uniformly mixing, centrifuging to obtain a supernatant, and transferring the supernatant into a new tube to obtain a purified solution; D. RNA isolation: adding DNaseI working mixed liquor into the purified liquor, uniformly mixing and incubating, adding the purified liquor 2, uniformly mixing and incubating, centrifuging and taking the supernatant to an RNase-free centrifugal tube to obtain RNA. The method has high dewaxing efficiency and no reagent residue problem, 1-3 tumor tissue slices can obtain enough RNA, the extracted RNA has high content and purity, the operation process is simple, convenient and quick, the repeatability is high, and the required sample amount is small.

Description

Method for rapidly extracting RNA (ribonucleic acid) of paraffin tissue

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a method for rapidly extracting RNA of paraffin tissue.

Background art:

the formalin-fixed paraffin-embedded tissue (FFPE, hereinafter referred to as FFPE) processing method can preserve tissues for a long time or prepare tissue specimens required for examination, and is the most commonly used method for preserving pathological tissues in medical institutions. A large amount of paraffin-embedded tissues accumulated in the files of the pathology department of the hospital and a huge amount of filed FFPE samples provide valuable resources for retrospective research, disease mechanism elucidation, treatment target discovery, prognosis indication and the like. Extraction of nucleic acids from archived FFPE tissue enables researchers to conduct various types of downstream research, including Polymerase Chain Reaction (PCR) -based DNA amplification diagnostics and retrospective molecular genetic studies, and therefore isolation of high purity, high quality nucleic acids from FFPE samples is the most fundamental prerequisite for successful downstream research.

However, extraction of high quality nucleic acids from FFPE tissue is difficult and challenging. 1) Formalin fixation and paraffin embedding of biological samples results in tissue nucleic acid damage, leading to extensive DNA and RNA fragmentation and unfavorable subsequent nucleic acid isolation. 2) At present, most of the conventional extraction methods are to dewax by using an organic solvent such as xylene, and then to purify by phenol chloroform extraction, ion exchange, salting out, silica gel column, etc.; but may interfere with downstream experiments due to dewaxing efficiency and reagent residue problems; and the obtained DNA and RNA have low purity, so that the requirements of downstream tests are difficult to meet. However, these methods usually only extract one kind of nucleic acid and waste the other kind of nucleic acid, and are time-consuming, require a large number of samples, and clinical molecular biology research usually requires simultaneous extraction of DNA and RNA, and when the samples are limited, the downstream test may be affected.

Therefore, a kit and a method for co-separating DNA and RNA from an FFPE sample are urgently needed in clinic, the purity of the DNA and RNA is high, and the yield is high, so that the normal operation of downstream tests can be ensured even under the condition of limited sample amount; and can ensure that the sources of DNA and RNA used in the research are completely the same.

The invention content is as follows:

the invention aims to provide a kit and a method for rapidly extracting RNA of paraffin tissue.

The kit for rapidly extracting RNA of paraffin tissue comprises the following components:

dewaxing mixed liquor buffer AL: 0.5-1.5% tween-20(V/V) and 5-15 mM Tris & Cl;

lysate buffer RLT: 400-600 mM Tris & Cl, 15-25 mM EDTA, 0.1-1% SDS and 80-120 mM DTT;

DNase I working mix: 8-12 mM Tris & Cl, 0.1Mm CaCl₂8-12 mM MnCl₂And DNaseI 0.5-7.5U/mu L;

purification solution 1: 1-5% Chelex-100(m/v, g/ml), and the solvent is TE buffer solution;

and (3) purifying liquid 2: 4-7% of Chelex-100(m/v, g/ml), and the solvent is DEPC treated water;

Proteinase K。

preferably, the dewaxed mixed liquor buffer AL contains 1% by volume of tween-20, 10mM Tris. Cl.

Preferably, the lysate buffer RLT comprises 500mM Tris. Cl, 20mM EDTA, 0.5% SDS by mass fraction, and 100mM DTT.

Preferably, the DNase I working mixture comprises 10mM Tris & Cl, 0.1mM CaCl₂，10mMMnCl₂、0.5U/μL DNase I。

Preferably, the purified solution 1 comprises 5% g/ml Chelex-100, and the solvent is TE buffer.

Preferably, the purified solution 2 comprises 6% g/ml Chelex-100, and the solvent is DEPC water.

The second purpose of the invention is to provide a method for rapidly extracting RNA of paraffin tissue, which comprises the following steps:

A. dewaxing: putting the FFPE sample into a dewaxing mixed liquor buffer AL for dewaxing;

B. lysis of the tissue: cracking the dewaxed sample by using a cracking solution buffer RLT and a protease K to obtain a cracking solution;

C. and (3) purification: adding the purified solution 1 into the lysate, uniformly mixing and incubating, centrifuging to obtain a supernatant, transferring the supernatant into a new tube, adding chloroform, uniformly mixing, centrifuging to obtain a supernatant, and transferring the supernatant into a new tube to obtain a purified solution;

D. RNA isolation: adding the DNase I working mixed solution into the purified solution, uniformly mixing and incubating, adding the purified solution 2, uniformly mixing and incubating, centrifuging and taking the supernatant to an RNase-free centrifuge tube to obtain RNA.

Preferably, the specific steps are as follows:

A. dewaxing:

scraping 1 piece of the paste with the thickness of 10 mu m and the area of about 1 multiplied by 1cm²Putting the FFPE sample into a 1.5mL centrifuge tube, adding 100 mul of dewaxing mixed solution buffer AL, immersing all tissues attached to the tube wall into the buffer AL, and incubating for 10min at 90 ℃;

B. lysis of tissue

(1) Adding 120 μ L lysis solution buffer RLT and 2ul protease K to make the final concentration of 10mg/L, vortexing, shaking, mixing uniformly, incubating at 56 deg.C for 15min 400rpm until the sample is completely dissolved, incubating at 80 deg.C for 15min, centrifuging, and collecting the solution on the tube wall to the tube bottom;

C. purification of

(1) Adding 100 μ l of purified solution 1, mixing, incubating at 99 deg.C 450rmp for 10min, immediately standing on ice for 2min, and centrifuging at 10500Xg for 15 min; (the paraffin yield was increased by placing the incubated sample on ice immediately after incubation and dewaxing was complete)

(2) Transferring the supernatant to a new EP tube, heating the supernatant to 45 ℃ in a metal bath, adding 100 mu l of chloroform, fully mixing the mixture, centrifuging the mixture for 15min at 10500Xg, and sucking 180 mu l of the supernatant into the new EP tube. (the upper phase is not sucked too much to avoid sucking the impurities in the middle phase and influencing the subsequent experiments.)

D. RNA isolation

(1) Adding 2 μ l of DNase I working mixture into the new EP tube containing the supernatant in step C (2), blowing and beating the mixture uniformly by using a pipette, incubating for 15min at 37 ℃, and incubating for 5min at 94 ℃;

(2) add 100. mu.l of purified solution 2, mix gently, incubate at 100 ℃ 450rmp for 15min, centrifuge at 10500Xg for 15min, transfer 180. mu.l of supernatant to a new RNase-free centrifuge tube.

The RNA isolated is recommended to be stored in a-80 ℃ refrigerator if it is not used immediately.

The method has high dewaxing efficiency and no reagent residue problem, 1-3 tumor tissue slices can obtain enough RNA, the extracted RNA has high content and purity, the operation process is simple, convenient and quick, the repeatability is high, and the required sample amount is small.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

RNA was isolated from 100 samples using a kit and compared in parallel with a conventional kit, and the samples were paraffin-embedded clinical specimens within 2 years.

1. Extraction reagent

① for RNA isolation

A. Dewaxing mixed liquor buffer AL: 1% tween-20(V/V), 10mM Tris & Cl, prepared by:

preparing 1M Tris & Cl: weighing 6.055g Tris, placing in a 100ml beaker, using DEPC water to fix the volume to 50ml, fully and uniformly mixing, and transferring to a glass reagent bottle without RNase and DNase for later use;

preparation of 10mM Tris. Cl: sucking 100ul 1M Tris & Cl, fixing the volume to 10ml with DEPC water, transferring to a glass reagent bottle without RNase and DNase, and fully and uniformly mixing for later use;

60ul of tween-20 is taken, the volume is adjusted to 6ml by 10mM Tris & Cl, and the dewaxing mixed liquor buffer AL is obtained by vortex, shaking and mixing.

B. Lysate buffer RLT: 500mM Tris & Cl, 20mM EDTA, 0.5% SDS by mass fraction and 100mM DTT, and the preparation method comprises the following steps:

configuration of 1M EDTA: weighing 14.612g of EDTA, placing in a 100ml beaker, using DEPC water to fix the volume to 50ml, fully and uniformly mixing, and transferring to a glass reagent bottle without RNase and DNase for later use;

configuration of 5% SDS: weighing 2.5g of SDS, placing in a 100ml beaker, using DEPC water to fix the volume to 50ml, fully and uniformly mixing, and transferring to a glass reagent bottle without RNase and DNase for later use;

configuration of 1M DTT: weighing 7.713g of DTT, placing the DTT in a 100ml beaker, using DEPC water to fix the volume to 50ml, fully and uniformly mixing, and transferring the mixture to a glass reagent bottle without RNase and DNase for later use;

preparation of lysate buffer RLT: 25ml of 1M Tris & Cl, 1ml of 1M EDTA, 5ml of 5% SDS and 5ml of 1M DTT are taken in a 100ml beaker, the volume is increased to 50ml by DEPC water, and the mixture is transferred to a glass reagent bottle without RNase and DNase for standby after being fully mixed.

C. DNase I working mix: 10mM Tris. Cl, 0.1mM CaCl₂，10mM MnCl₂0.5U/. mu.L DNaseI, and the preparation method comprises the following steps:

10mM of CaCl₂The configuration of (2): weighing 0.0555gCaCl₂Placing in a 100ml beaker, fixing the volume to 50ml with DEPC water, transferring to a glass reagent bottle without RNase and DNase, and fully shaking for later use;

100mM MnCl₂The configuration of (2): weighing 0.629g of MnCl₂Placing in a 100ml beaker, fixing the volume to 50ml with DEPC water, transferring to a glass reagent bottle without RNase and DNase, and fully shaking for later use;

configuration of DNase I buffer: 100ul 100mM Tris & Cl, 10ul 10mM CaCl₂、100ul100mM MnCl₂Transferring to a centrifugal tube without RNase and DNase, adding 790ul DEPC water, fixing the volume to 1ml, and uniformly mixing by vortex oscillation for later use;

preparing DNase I working mixed liquid: DNase I buffer and 1U/. mu.LDNase I are used according to the volume ratio of 1: 1.

C. Purification solution 1: 5% Chelex-100(m/v, g/ml) is dissolved in TE buffer solution, and the preparation method is as follows: weighing 0.5g of Chelex-100 in a reagent bottle of RNase and DNase, adding 10ml of TE buffer solution, and shaking gently before use;

D. and (3) purifying liquid 2: 6% Chelex-100(m/v, g/ml) is dissolved in DEPC water and is prepared by the following method: weighing 0.6g of Chelex-100 into a reagent bottle of RNase and DNase, adding 10ml of DEPC water, and shaking gently before use;

2. extraction method

Step 1 dewaxing

(1) Scraping 1 piece of the paste with the thickness of 10 mu m and the area of about 1 multiplied by 1cm²The FFPE sample is put into a 1.5mL centrifuge tube, and 100. mu.l of dewaxing mixed solution buffer is addedAL, immersing all tissues attached to the tube wall in the AL, and incubating for 10min at 90 ℃;

step 2 lysis of tissue

(1) Then 120. mu.l of lysate buffer RLT and 2ul of protease K (to a final concentration of 10mg/L) were added, vortexed, mixed, incubated at 56 ℃ for 15min and 400rpm until the sample was completely dissolved, and incubated at 80 ℃ for 15 min. Centrifuging for a short time to collect the solution on the tube wall to the tube bottom;

step 3, purification

(1) Adding 100 μ l of purified solution 1 into the tube, mixing, incubating at 99 deg.C and 450rpm for 10min, immediately standing on ice for 2min, and centrifuging at 10500 × g for 15 min; (the paraffin yield was increased by placing the incubated sample on ice immediately after incubation and dewaxing was complete)

(2) Transferring the supernatant to a new EP tube, heating to 45 deg.C with metal bath, adding 100 μ l chloroform, mixing, centrifuging at 10500 × g for 15min, and sucking 180 μ l supernatant into the new EP tube. (the upper phase is not sucked too much to avoid sucking the impurities in the middle phase and influencing the subsequent experiments.)

Step 4.RNA isolation

(1) Adding 2 mul of DNase I working mixed solution into the EP tube in the previous step, blowing and beating the mixture uniformly by using a pipette, incubating the mixture for 15min at 37 ℃, and incubating the mixture for 5min at 94 ℃;

(2) adding 100 μ l of purified solution 2, mixing, incubating at 100 deg.C and 450rpm for 15min, and centrifuging at 10500 × g for 15 min; transfer 180. mu.l of the supernatant to a new RNase-free centrifuge tube to obtain RNA.

The RNA isolated is recommended to be stored in a-80 ℃ refrigerator if it is not used immediately.

3. Detection of the RNA content

The RNA extracted by the kit is subjected to content value detection by adopting the Qubit4,

the results are shown in Table 1:

TABLE 1 RNA content extracted by the method of the invention and by the usual kit

The result shows that the yield of the nucleic acid extracted by the kit is obviously higher than that of the nucleic acid extracted by the common centrifugal column method, and the quality and the purity of the nucleic acid are both better than those of the nucleic acid extracted by the common centrifugal column method.

Comparative example 1:

this comparative example is essentially the same as example 1 except that xylene was used in place of dewaxed blend AL for dewaxing and the FFPE samples were of different sizes.

The results are shown in Table 3 and Table 3

As can be seen from Table 3, the substitution of xylene with Tween (dewaxed mixed solution buffer AL) is effective in improving the concentration and quality (purity) of RNA.

Comparative example 2:

this comparative example is essentially the same as example 1 except that xylene was lysed with normal buffer (50mM Tri-hcl, 20mM EDTA, 100mM NaCl, pH7.5) instead of the lysate buffer RLT for deparaffinization, and the FFPE samples were of different sizes.

The results are shown in Table 4

TABLE 4

Measured value (mean value)	Concentration (ng/. mu.l)	A260/A280	A260/A230
				Lysis solution bufferrLT	346	2.01	2.60
Lysis with common buffer	245	1.76	2.11

As can be seen from Table 4, the substitution of xylene with Tween (dewaxed mixed solution buffer AL) is effective in improving the concentration and quality (purity) of RNA.

Comparative example 3:

this comparative example is essentially the same as example 1 except that conventional column purification was used for dewaxing instead of purified liquor 1 and purified liquor 2, and the FFPE samples were of different sizes.

The results are shown in Table 5

TABLE 5

Measured value (mean value)	Concentration (ng/. mu.l)	A260/A280	A260/A230
				Purification solutions 1 and 2 of the invention	368	2.02	2.54
Conventional column purification	257	1.82	2.15

As can be seen from Table 5, the replacement of conventional column chromatography with Chelex-100 effectively improved the concentration and quality (purity) of RNA.

Example 2:

this example is essentially the same as example 1, except that the extraction reagents are different as follows:

dewaxing mixed liquor buffer AL: 0.5% tween-20(V/V), 5mM Tris & Cl;

lysate buffer RLT: 400mM Tris & Cl, 15mM EDTA, 0.1% SDS, 80mM DTT and 10mg/L Proteinase K;

DNase I working mix: 8mM Tris. Cl, 0.1Mm CaCl₂8mM MnCl₂And DNase I0.5U/. mu.L;

purification solution 1: 1% Chelex-100(m/v, g/ml), and TE buffer as solvent;

and (3) purifying liquid 2: 4% Chelex-100(m/v, g/ml) and the solvent is DEPC treated water.

Example 3:

this example is essentially the same as example 1, except that the extraction reagents are different as follows:

dewaxing mixed liquor buffer AL: 1.5% tween-20(V/V), 15mM Tris. Cl;

lysate buffer RLT: 600mM Tris & Cl, 25mM EDTA, 1% SDS by mass fraction, 120mM DTT;

DNase I working mix: 12mM Tris. Cl, 0.1Mm CaCl₂12mM MnCl₂And DNase I7.5U/. mu.L;

purification solution 1: 5% Chelex-100(m/v, g/ml), and TE buffer as solvent;

and (3) purifying liquid 2: 7% Chelex-100(m/v, g/ml) and the solvent is DEPC treated water.

Claims (8)

1. A kit for rapidly extracting RNA from paraffin tissue is characterized by comprising the following components:

dewaxing mixed liquor buffer AL: 0.5-1.5% tween-20(V/V) and 5-15 mM Tris & Cl;

lysate buffer RLT: 400-600 mM Tris & Cl, 15-25 mM EDTA, 0.1-1% SDS and 80-120 mM DTT;

DNase I working mix: 8-12 mM Tris & Cl, 0.1Mm CaCl₂8-12 mM MnCl₂And DNase I0.5-7.5U/mu L;

purification solution 1: 1-5% Chelex-100(m/v, g/ml), and the solvent is TE buffer solution;

and (3) purifying liquid 2: 4-7% of Chelex-100(m/v, g/ml), and the solvent is DEPC treated water;

proteinase K.

2. The kit of claim 1, wherein the dewaxed mixed solution buffer comprises a volume fraction of 1% tween-20, 10mM Tris-Cl.

3. The kit of claim 1, wherein the lysis buffer RLT comprises 500mM Tris-Cl, 20mM EDTA, 0.5% SDS by mass fraction, and 100mM DTT.

4. The kit of claim 1, wherein the DNase I working mixture comprises 10mM Tris-Cl, 0.1mM CaCl₂，10mM MnCl₂、0.5U/μLDNase I。

5. The kit of claim 1, wherein the purified solution 1 comprises 5% g/ml of Chelex-100, and the solvent is TE buffer.

6. The kit of claim 1, wherein the purified solution 2 comprises 6% g/ml of Chelex-100 and the solvent is DEPC water.

7. A method for rapidly extracting RNA from paraffin tissue, which is characterized in that the kit of claim 1, 2, 3, 4, 5 or 6 is used for extraction, and the specific method is as follows:

A. dewaxing: putting the FFPE sample into a dewaxing mixed liquor buffer for dewaxing;

B. lysis of the tissue: cracking the dewaxed sample by using a cracking solution buffer RLT and protease K to obtain a cracking solution;

C. and (3) purification: adding the purified solution 1 into the lysate, uniformly mixing and incubating, centrifuging to obtain a supernatant, transferring the supernatant into a new tube, adding chloroform, uniformly mixing, centrifuging to obtain a supernatant, and transferring the supernatant into a new tube to obtain a purified solution;

D. RNA isolation: adding the DNase I working mixed solution into the purified solution, uniformly mixing and incubating, adding the purified solution 2, uniformly mixing and incubating, centrifuging and taking the supernatant to an RNase-free centrifuge tube to obtain RNA.

8. The method according to claim 7, characterized by the following specific steps:

A. dewaxing:

scraping 1 piece of the paste with the thickness of 10 mu m and the area of about 1 multiplied by 1cm²Putting the FFPE sample into a 1.5mL centrifuge tube, adding 100 mu l of dewaxing mixed solution buffer AL, immersing all tissues attached to the tube wall into the FFPE sample, and incubating for 10min at 90 ℃;

B. lysis of tissue

(1) Adding 120 mul of lysis buffer RLT and 2 mul of protease K to enable the final concentration to be 10mg/L, whirling, shaking, uniformly mixing, incubating at 56 ℃ for 15min and 400rpm until the sample is completely dissolved, incubating at 80 ℃ for 15min, and centrifuging to enable the solution on the tube wall to be collected to the tube bottom;

C. purification of

(1) Adding 100 μ l of purified solution 1, mixing, incubating at 99 deg.C 450rmp for 10min, immediately standing on ice for 2min, and centrifuging at 10500Xg for 15 min;

(2) transferring the supernatant to a new EP tube, heating the supernatant to 45 ℃ in a metal bath, adding 100 mu l of chloroform, fully mixing, centrifuging for 15min at 10500Xg, and sucking 180 mu l of the supernatant into the new EP tube;

D. RNA isolation

(1) Adding 2 μ l of DNase I working mixture into the new EP tube containing the supernatant in step C (2), blowing and beating the mixture uniformly by using a pipette, incubating for 15min at 37 ℃, and incubating for 5min at 94 ℃;

(2) add 100. mu.l of purified solution 2, mix gently, incubate at 100 ℃ 450rmp for 15min, centrifuge at 10500Xg for 15min, transfer 180. mu.l of supernatant to a new RNase-free centrifuge tube.