CN105713963A - Method for detecting gene expression in formalin fixed and paraffin embedded tissue sample - Google Patents
Method for detecting gene expression in formalin fixed and paraffin embedded tissue sample Download PDFInfo
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- CN105713963A CN105713963A CN201410737231.0A CN201410737231A CN105713963A CN 105713963 A CN105713963 A CN 105713963A CN 201410737231 A CN201410737231 A CN 201410737231A CN 105713963 A CN105713963 A CN 105713963A
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Abstract
The invention discloses a method for detecting gene expression in a formalin fixed and paraffin embedded tissue sample. The method comprises the following steps of treating the formalin fixed and paraffin embedded tissue sample by dewaxing liquid to obtain a dewaxed tissue sample; cracking the dewaxed tissue sample to obtain a tissue sample cracking solution; and directly detecting the gene expression in the cracking solution.
Description
Invention field
The method that the present invention relates to detection gene expression, particularly to fixing the method quickly detecting gene expression in paraffin-embedded tissue sample from formalin.
Background technology
The announcement of human genome DNA's complete sequence data indicates that life sciences development has stepped into the genomics epoch.Along with molecular biological abundant development, with nucleic acid be main study subject Protocols in Molecular Biology more and more widely by the mankind field for medical diagnosis on disease, for disease prevention, predict, offer information and decision-making foundation be provided.
Formalin fixes paraffin-embedded tissue (formalinfixedandparaffinembeddedtissues is called for short FFPE) as the most frequently used method preserving pathological tissue of medical institutions, is the material source of a reliable molecular biology research.Owing to fresh specimens is difficult to obtain, for carrying out entity tumor molecular biology research, when existing case is carried out retrospective study, can only carrying out gene extraction from paraffin-embedded tissue, therefore paraffin-embedded tissue just becomes source the abundantest precious resources.Gene expression in detection FFPE sample at present needs to isolate high-quality RNA from FFPE sample.But, due to RNA Partial digestion in FFPE sample, and often closely cross-link with histone, therefore want to extract information nucleic acid therein quite difficult.The at present conventional method length consuming time extracting total serum IgE from FFPE sample, and numerous and diverse purification step causes a large amount of losses of RNA sample, when sample size has in limited time, is then likely to affect the carrying out of detected downstream.Accordingly, it would be desirable to exploitation is a kind of simple, quickly, method detects the expression of gene from FFPE sample accurately.
Summary of the invention
One aspect of the application fixes the quick method detecting gene expression in paraffin-embedded tissue sample particularly to from formalin, including: formalin is fixed paraffin-embedded tissue sample dewaxing liquid and is processed to obtain the tissue sample of dewaxing by (a)；B the tissue sample of described dewaxing is cracked to obtain tissue sample lysate by ()；(c) gene expression in described lysate is directly detected.
In some embodiments, step (c) includes the mRNA that directly detects the described gene in described lysate.
In some embodiments, step (c) including: provides the probe pair that is made up of reporter probe and capture probe, the first nucleotide sequence that the mRNA that wherein said reporter probe comprises (1) and described gene is hybridized；(2) the fluorescent labeling bar code on described first nucleotide sequence it is connected to；The second nucleotide sequence that the mRNA that wherein said capture probe comprises (1) and described gene is hybridized；(2) biotin on described second nucleotide sequence it is connected to；The mRNA of described probe pair with described gene is hybridized and forms complex；Described complex is fixed on the substrate with Avidin with the state extended；The signal-count of detection fluorescent labeling bar code.
In some embodiments, use nCounter to analyze system in step (c) and detect the gene expression of described lysate.
In some embodiments, paraffin-embedded tissue sample fixed by described formalin is microcomponent's sample.In certain embodiments, paraffin-embedded tissue sample fixed by described formalin is fine needle aspiration biopsy sample.In certain embodiments, described formalin fixes the volume of paraffin-embedded sample more than 0.75 × 10-2mm3.In certain embodiments, described formalin fixes the volume of paraffin-embedded sample more than 1.5 × 10-2mm3.In certain embodiments, described formalin fixes the volume of paraffin-embedded sample more than 3 × 10-2mm3.In certain embodiments, described formalin fixes the volume of paraffin-embedded sample more than 6 × 10-2mm3.In certain embodiments, described formalin fixes the volume of paraffin-embedded sample more than 12 × 10-2mm3。
In some embodiments, described dewaxing liquid comprises dimethylbenzene.
In some embodiments, protease k is used to be cracked by the tissue sample of described dewaxing in step (b).
In some embodiments, described lysate was used desoxyribose ferment treatment before step (c).
In some embodiments, paraffin-embedded tissue sample fixed by described formalin is people's tissue sample.In some embodiments, described people's tissue sample is derived from the pathologic tissue samples of patient.In some embodiments, described pathologic tissue samples is neoplasmic tissue sample.In some embodiments, described tumor tissues derives from the tumor selected from lower group: minicell or lung cancer in non-cellule type, gastric cancer, intestinal cancer, esophageal carcinoma, colorectal carcinoma, carcinoma of prostate, ovarian cancer, breast carcinoma, the brain cancer, urinary tract cancer, renal carcinoma, bladder cancer, malignant melanoma, hepatocarcinoma, uterus carcinoma, cancer of pancreas, myeloma cancer, carcinoma of endometrium, head and neck cancer, tumors in children, sarcoma.
In some embodiments, described gene is ALK.
The brief description of accompanying drawing
Fig. 1. from xenotransplantation FFPE tissue sample, quickly detect expression conditions.For the same gene (ALK) of same tissue sample, directly detect the count signal obtained with lysate and be about detect the signal obtained with the total serum IgE of purification 2 times.
Fig. 2. detect expression conditions with lysate.A large amount of on pulmonary carcinoma adenocarcinoma clinical sample repeat lysates preparation experiment, and compare with the total serum IgE experimental result of 500ng purification, result show both the very high (R of concordance2=0.946).
Fig. 3. detect the expression conditions in microcomponent's sample with lysate.The total serum IgE (using RNeasyFFPEKit (Qiagen)) of lysate and purification is prepared from the FFPE of equal area, same sample organizing.When tissue sample volume is about 6 × 10-2mm3Time, use the signal that obtains of the total serum IgE threshold value (60) close to background signal of purification, it is impossible to draw metrical information accurately.When tissue sample volume is less than 3 × 10-2mm3, use the signal that obtains of the total serum IgE threshold value lower than background signal of purification.And can accurately detect volume with lysate is 1.5 × 10-2mm3FFPE tissue sample.
Detailed Description Of The Invention
This application discloses a kind of method fixing quickly detection gene expression paraffin-embedded tissue sample from formalin, comprise the following steps:
A formalin is fixed paraffin-embedded tissue sample dewaxing liquid and is processed to obtain the tissue sample of dewaxing by ()；
B the tissue sample of described dewaxing is cracked to obtain tissue sample lysate by ()；With
C () directly detects the gene expression in described lysate.
In this application, detection gene expression refers to the amount detecting the corresponding gene outcome produced by the DNA sequence of gene, for instance the amount of mRNA.In some embodiments, detection gene expression includes the DNA sequence of detection gene and transcribes the amount of the mRNA obtained.Owing to the shear history after rna transcription is likely to different, same gene is likely to one or more different mRNA transcriptons corresponding.In some embodiments, detection gene expression is the amount of detection a certain kind of answering of gene pairs or several mRNA transcripton with particular sequence.
In this application, " paraffin embedding fixed by formalin " refers to and fixes with suitable chemical reagent, dehydration the biological sample infected by paraffin.In fixation procedure, cell component quick solidification in biological sample and crosslinking, thus being able to long-term preservation.Formalin (formaldehyde), ethanol, acetic acid, mercuric chloride, osmic acid (Osmic acid .), potassium dichromate and picric acid or its combination is included but not limited to for fixing chemical reagent.Dehydration can include carrying out dehydration step by step with the ethanol (such as 30%, 50%, 70%, 80%, 90% to 100%) of variable concentrations successively.Other dehydrant includes but not limited to n-butyl alcohol, the tert-butyl alcohol, acetone.Paraffin infects and the process that embeds is known in this field.
In this application, the tissue sample for detecting can include animal tissue's sample and Plant tissue samples.In some embodiments, tissue sample is people's tissue sample, it is preferred that be derived from the pathologic tissue samples of patient.According to some embodiment, tissue sample is neoplasmic tissue sample.According to some embodiment, tumor tissues derives from the tumor selected from lower group: minicell or lung cancer in non-cellule type, gastric cancer, intestinal cancer, esophageal carcinoma, colorectal carcinoma, carcinoma of prostate, ovarian cancer, breast carcinoma, the brain cancer, urinary tract cancer, renal carcinoma, bladder cancer, malignant melanoma, hepatocarcinoma, uterus carcinoma, cancer of pancreas, myeloma cancer, carcinoma of endometrium, head and neck cancer, tumors in children, sarcoma.
In this application, the detection of gene expression refers to and detects this DNA sequence and have bioactive product in tissue sample by detecting the quantity of RNA encoded by specific dna sequence, including the amount of mRNA and protein molecular.In some embodiments, the detection of gene expression refers to the mRNA fragment measuring specific gene.
In this application, the paraffin that dewaxing liquid refers to be fixed by formalin in paraffin-embedded tissue sample removes simultaneously chemical reagent or the solution of not lesion tissue sample.In some embodiments, dewaxing liquid comprises dimethylbenzene.In some embodiments, dewaxing liquid can be the reagent bought on market, for instance the DeparaffinizationSolution of Kai Jie company (Qiagen).
In this application, refer to the step and process that make FFPE tissue sample dewax with dewaxing liquid with dewaxing liquid " process " FFPE tissue sample, mix including by dewaxing liquid and FFPE tissue sample.According to some embodiment, " process " can also include by dewaxing liquid with dewaxing after tissue sample separate.According to some embodiment, process FFPE tissue sample with dewaxing liquid to include in test tube, mix dewaxing liquid and FFPE tissue sample, the tissue sample of centrifugation dewaxing liquid and dewaxing, remove dewaxing liquid, test tube adds ethanol purge tissue sample to remove the dewaxing liquid of residual, by centrifugal and evaporating ethanol, thus obtaining the tissue sample of dewaxing.
In this application, " tissue sample of dewaxing " refers to that the tissue sample that paraffin-embedded tissue sample eliminates more than 60% after processing through dewaxing liquid, obtains after the paraffin of 70%, 80%, 90% to 100% fixed by formalin.
In this application, " cracking " refers to that the integrity of the membrane structure in tissue sample is destroyed, and makes intra-cellular components, process out as free in albumen, DNA and RNA etc..According to some embodiment, by processing tissue sample with E.C. 3.4.21.64, tissue sample is cracked.
" lysate " refer to tissue sample cleaved after the original stock of free cell component that obtains.Lysate can be further processed with separation, purification or expand certain component therein.In some embodiments, the tissue sample lysate used in method described herein is not past any process carried out for the purpose of separation, purification or amplification wherein certain component, or the process not past the separation that RNA therein is carried out, purification or amplification.In some embodiments, alternatively, lysate with desoxyribose ferment treatment to remove the DNA in lysate.In this application, the direct detection to gene expression is not affected with desoxyribose ferment treatment.
In this application, " directly " refer to detect gene expression time, the component in lysate is not past separation, purification or amplification.Such as, the RNA in lysate does not have separated, purification or amplification, for instance do not use silicon dioxide column purification, also without by methods such as such as PCR, RNA reverse transcription becomes cDNA and/or amplification.Detect the gene expression in paraffin-embedded sample by prior art, be typically necessary from sample and separate purifying RNA, and the RNA reverse transcription of purification is become cDNA and uses pcr amplification.Such method formality is loaded down with trivial details, length consuming time, and accurate not.Particularly in, in the less situation of sample size, due to sample loss inevitable in the process of separation purifying RNA, causing the gene expression that prior art cannot accurately detect in sample.By directly detecting the gene expression in lysate, this method can reduce the process step to lysate thus reducing the loss of RNA, can also avoid because the amplification method such as PCR processes the error brought, thus in the shorter time expression conditions of Accurate Determining tissue sample.
In some embodiments, directly detecting the gene expression in lysate can by using the method for numeral multi-gene expression analysis (Digitalmultiplexedgeneexpressionanalysis) be analyzed.Numeral multi-gene expression analysis refers to and a kind of utilizes molecular barcode and single molecular imaging to detect and add up the method for the quantity of particular transcripts in each reaction system, its principle can referring to Geiss etc., Directmultiplexedmeasurementofgeneexpressionwithcolor-co dedprobepairs, NatureBiotechnology, 26:317-325；U.S. Patent application US20100015607A1.The method includes providing the probe pair that is made up of reporter probe and capture probe, the first nucleotide sequence that the mRNA that wherein said reporter probe comprises (1) and described gene is hybridized；(2) it is connected to the fluorescent labeling bar code that described first nucleotide sequence 5 ' is held；The second nucleotide sequence that the mRNA that wherein said capture probe comprises (1) and described gene is hybridized；(2) it is connected to the biotin that described second nucleotide sequence 3 ' is held；The mRNA of described probe pair with described gene is hybridized and forms complex；Described complex is fixed on the substrate with Avidin with the state extended；The signal-count of detection fluorescent labeling bar code.
In certain embodiments, directly detecting the gene expression in lysate can by using nCounter analysis system complete.In simple terms, nCounter analysis system adopts the probe that two length are about 50bp to hybridize with mRNA.Reporter probe (reporterprobe) carries signal；Capture probe (captureprobe) is fixed for the complex after hybridizing, for data acquisition.After hybridization, sample is transferred to sample preparation effort station, and probe excessive there is washed off, and the complex of probe and purpose mRNA is then fixed on the specimen holder of nCounter.Specimen holder is placed on data analyzer and carries out data acquisition.Color coded numbers different on specimen holder will be computed, and represent the quantity of each molecules of interest.In a particular embodiment, nCounter analyzes system and is produced by NanoString company of the U.S., including full automatic sample treatment work station, digitalized image analyser, CodeSet (molecular barcode) and related reagent.This system is used to carry out detecting without using enzyme, it is not necessary to reverse transcription, it is not required that do pcr amplification.(referring to Kulkarni, DigitalmultiplexedgeneexpressionanalysisusingtheNanoStri ngnCountersystem.)
The lysate of FFPE tissue sample is analyzed method detection gene expression either directly through numeral multi-gene expression by disclosed method.Compared with the method needing RNA purification, the present processes avoids and lysate is repeatedly processed, and greatly reduces RNA loss, thus saving time and required sample, reducing cost simultaneously, saving reagent and consumptive material consumption.Especially, disclosed method is owing to having only to minimal amount of tissue sample, thus is applicable to the tissue sample that area is small, for instance fine needle aspiration biopsy sample (fine-needlebiopsy).Simultaneously as disclosed method is simple to operate, therefore easily realize automation mechanized operation.By embodiment, the application shows that in the lysate prepared by disclosed method, RNA mass is intact, it does not have obvious degradation phenomenon.And the present processes is compared with the testing result of the RNA of use purification, both concordance in qualitative results are very high, but the amount of the RNA that the present processes records is significantly larger than the amount that the detection method of the RNA using purification records, this shows that the present processes avoids a large amount of losses of RNA in sample
According to some embodiment, formalin is fixed paraffin-embedded tissue sample and is prepared by fine needle aspiration biopsy sample (fine-needlebiopsies)." fine needle aspiration biopsy " refers to a kind of technology detecting subcutaneous lump or lump.This technology carries out cell sampling by tiny hollow needle is inserted lump, in conjunction with dyeing and molecular diagnosis, it is possible to avoid operation sampling detection.At present, Pintle puncture technology is widely used in the diagnosis of cancer and immunological diseases.
Disclosed method is particularly well-suited to from the FFPE tissue sample of trace to detect gene expression.According to some embodiment, formalin fixes the volume of paraffin-embedded sample more than 0.75 × 10-2mm3、1.0×10-2mm3、1.5×10-2mm3、2×10-2mm3、2.5×10-2mm3、3×10-2mm3、3.5×10-2mm3、4×10-2mm3、4.5×10-2mm3、5×10-2mm3、6×10-2mm3、7×10-2mm3、8×10-2mm3、9×10-2mm3、10×10-2mm3、11×10-2mm3、12×10-2mm3、13×10-2mm3、14×10-2mm3、15×10-2mm3.According to some embodiment, the volume of paraffin-embedded sample fixed by formalin is 1.5～6 × 10-2mm3.According to some embodiment, the volume of paraffin-embedded sample fixed by formalin is 1.5～3 × 10-2mm3。
Embodiment
Embodiment 1. quickly detects expression conditions from xenotransplantation FFPE tissue sample
Material:
Adenocarcinoma FFPE tissue sample is from Korea S.Tumor xenograft FFPE tissue samples is from the bright Kant of medicine.
Method:
1, cut the tissue slice of 4-5 μ m-thick with scalpel, put in 1.5ml centrifuge tube.
2, described centrifuge tube adds 1ml dimethylbenzene, vortex mixed 10 seconds, is centrifuged 4 minutes under 20,000 × g.
3, remove supernatant carefully, retain precipitate.
4, precipitate adds 1 milliliter of 96-100% ethanol, vortex mixed, is centrifuged 2 minutes under 20,000 × g.
5, it is carefully removed supernatant, removes the ethanol of residual when interference precipitation as far as possible.
6, at room temperature air drying, it is ensured that all of ethanol evaporation.
7, the precipitation dried with appropriate proteinase K buffer resuspension, add 5 μ L E.C. 3.4.21.64s (20 mg/ml).
8, react 15 minutes at 56 DEG C, then place 15 minutes at 80 DEG C.
9, it is centrifuged 10-15 minute under 20,000 × g.
10, supernatant is moved on to a new 1.5ml centrifuge tube and obtains lysate.
11, analyze the gene expression in system detection lysate with nCounter.
As a comparison, the FFPERNA purification kit (RNeasyFFPEKit) produced with Kai Jie company (Qiagen) prepares total serum IgE from same tissue samples, and analyze the gene expression of system detection total serum IgE with nCounter
Result:
As it is shown in figure 1, for the same gene (ALK) of same tissue sample, directly detect the count signal obtained with lysate and be about detect the signal obtained with the total serum IgE of purification 2 times.
Embodiment 2. detects expression conditions with lysate
Material: adenocarcinoma FFPE tissue sample is from Korea S.
Method:
Pulmonary carcinoma adenocarcinoma clinical sample repeats in a large number detect gene expression experiment with lysate.
1, cut the tissue slice of 5 μ m-thick with scalpel, put in 1.5ml centrifuge tube.
2, described centrifuge tube adds 1ml dimethylbenzene, vortex mixed 10 seconds, is centrifuged 4 minutes under 20,000 × g.
3, remove supernatant carefully, retain precipitate.
4, precipitate adds 1 milliliter of 96-100% ethanol, vortex mixed, is centrifuged 2 minutes under 20,000 × g.
5, it is carefully removed supernatant, removes the ethanol of residual when interference precipitation as far as possible.
6, at room temperature air drying, it is ensured that all of ethanol evaporation.
7, the precipitation dried with appropriate proteinase K buffer resuspension, add 5 μ L E.C. 3.4.21.64s (20 mg/ml).
8, react 15 minutes at 56 DEG C, then place 15 minutes at 80 DEG C.
9, it is centrifuged 10-15 minute under 20,000 × g.
10, supernatant is moved on to a new 1.5ml centrifuge tube and obtains lysate.
11, lysate adds buffer and the DNA enzymatic of the DNA enzymatic being equivalent to total amount of liquid 1/10.At room temperature hatch 15 minutes, then place 15 minutes at 70 DEG C, and in cooled on ice.
12, analyze the gene expression in system detection lysate with nCounter.
As a comparison, the FFPERNA purification kit (RNeasyFFPEKit) produced with Kai Jie company (Qiagen) prepares total serum IgE from same tissue samples, and analyze the gene expression of the system detection 500ng total serum IgE purified with nCounter.
Result:
As in figure 2 it is shown, detect gene expression and the very high (R of result concordance detected with the total serum IgE of purification with lysate2=0.946).
Embodiment 3. lysate detects the expression conditions in microcomponent's sample
For more small and precious FFPE tissue (such as the tissue of fine-needlebiopsy similar size), compare target gene (ALK, ROS1 and RET) mrna expression amount in the total serum IgE of lysate and the purification prepared from equivalent sample.Prepare the total serum IgE (using RNeasyFFPEKit (Qiagen)) of lysate and purification organizing from the FFPE of equal area, same sample, carry out the mensuration of gene expression respectively.Result is as it is shown on figure 3, common total RNA extraction reagent box have lost the RNA of about 75%-88% in purification step.When tissue sample volume is about 6 × 10-2mm3Time, use the signal that obtains of the total serum IgE threshold value (60) close to background signal of purification, it is impossible to draw metrical information accurately.When tissue sample volume is less than 3 × 10-2mm3, use the signal that obtains of the total serum IgE threshold value lower than background signal of purification.And can accurately detect volume 1.5～6 × 10 with lysate-2mm3FFPE tissue sample.
Claims (15)
1. fix, from formalin, the method quickly detecting gene expression paraffin-embedded tissue sample, comprise the following steps:
A formalin is fixed paraffin-embedded tissue sample dewaxing liquid and is processed to obtain the tissue sample of dewaxing by ()；
B the tissue sample of described dewaxing is cracked to obtain tissue sample lysate by ()；With
C () directly detects the gene expression in described lysate.
2. the method for claim 1, it is characterised in that step (c) includes the mRNA directly detecting the described gene in described lysate.
3. method as claimed in claim 2, it is characterised in that step (c) includes
The probe pair being made up of reporter probe and capture probe is provided,
The first nucleotide sequence that the mRNA that wherein said reporter probe comprises (1) and described gene is hybridized；(2) the fluorescent labeling bar code on described first nucleotide sequence it is connected to；
The second nucleotide sequence that the mRNA that wherein said capture probe comprises (1) and described gene is hybridized；(2) biotin on described second nucleotide sequence it is connected to；
The mRNA of described probe pair with described gene is hybridized and forms complex；
Described complex is fixed on the substrate with Avidin with the state extended；
The signal-count of detection fluorescent labeling bar code.
4. method as claimed in claim 3, it is characterised in that use nCounter to analyze system in step (c) and detect the gene expression of described lysate.
5. the method for claim 1, it is characterised in that paraffin-embedded tissue sample fixed by described formalin is fine needle aspiration biopsy sample.
6. the method for claim 1, it is characterised in that described formalin fixes the volume of paraffin-embedded sample more than 0.75 × 10-2mm3。
7. the method for claim 1, it is characterised in that described formalin fixes the volume of paraffin-embedded sample more than 1.5 × 10-2mm3。
8. the method for claim 1, it is characterised in that described dewaxing liquid comprises dimethylbenzene.
9. the method for claim 1, it is characterised in that use protease k to be cracked by the tissue sample of described dewaxing in step (b).
10. the method for claim 1, it is characterised in that described lysate was used desoxyribose ferment treatment before step (c).
11. the method for claim 1, it is characterised in that paraffin-embedded tissue sample fixed by described formalin is people's tissue sample.
12. method as claimed in claim 11, it is characterised in that described people's tissue sample is derived from the pathologic tissue samples of patient.
13. method as claimed in claim 12, it is characterised in that described pathologic tissue samples is neoplasmic tissue sample.
14. method as claimed in claim 13, it is characterized in that, described tumor tissues derives from the tumor selected from lower group: minicell or lung cancer in non-cellule type, gastric cancer, intestinal cancer, esophageal carcinoma, colorectal carcinoma, carcinoma of prostate, ovarian cancer, breast carcinoma, the brain cancer, urinary tract cancer, renal carcinoma, bladder cancer, malignant melanoma, hepatocarcinoma, uterus carcinoma, cancer of pancreas, myeloma cancer, carcinoma of endometrium, head and neck cancer, tumors in children, sarcoma.
15. the method for claim 1, it is characterised in that described gene is selected from lower group: ALK, ROS1 and RET.
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