Eluent for removing fluorescence detection probe, elution method and RNA (Ribonucleic Acid) in-situ detection method

Abstract

The invention relates to the technical field of nucleic acid in-situ detection, in particular to an eluent for removing a fluorescence detection probe, an elution method and an RNA in-situ detection method. The technical scheme provided by the invention is used for removing the fluorescent detection probe with the length of 20-30bp in multiple rounds of detection, and the fluorescent detection probe for in-situ sequencing can be quickly and efficiently removed; only incubation at 37 DEG C is needed for elution, and the eluent is neutral, so that the tissue is not damaged, and the tissue form is kept intact; a background is not introduced, so that the tissue background generated by multiple times of elution can be reduced, and the reaction time of the whole process is shortened. In the related technology, a detection probe elution strategy is long in clearing time or high in clearing frequency, a certain background is introduced, fluorescence imaging of subsequent rounds is affected, the signal-to-noise ratio is reduced, the signal detection amount of the method is reduced, and meanwhile, due to introduction of the background, the detectable target flux of an in-situ sequencing method is limited.

Description

Technical Field

[0001] The present application relates to the technical field of nucleic acid in situ detection, and in particular to an elution solution and an elution method for removing fluorescent detection probes, and an RNA in situ detection method. Background Art

[0002] The key to RNA in situ sequencing technology lies in the elution of the detection probe and the re-incubation detection of the next round of sequencing probe. Under the action of DNA ligase, the fluorescent detection probe is connected to the anchor sequence to form a detection probe with a sequence length of 20 to 30 bp, which is firmly bound to the rolling circle amplification product to complete a complete round of sequencing. Between the two rounds of sequencing steps, the detection probe of the previous round needs to be completely removed from the rolling circle amplification product to fully expose the site on the product for binding to the next round of detection probe for the next round of sequencing.

[0003] Usually, the strategy for eluting the detection probe in the previous round is to use high-concentration formamide for incubation and removal, or to use low-concentration reaction solution for long-term incubation and removal. However, these commonly used removal schemes may cause tissue damage, resulting in severe tissue fluorescence background or requiring a long time to achieve effective removal.

[0004] Therefore, there is an urgent need for a solution that causes little damage to tissues and can quickly remove the detection probes. Summary of the invention

[0005] The present application provides an elution solution, an elution method, and an RNA in situ detection method for removing a fluorescent detection probe.

[0006] The technical solution provided in the present application is used to remove fluorescent detection probes with a length of 20 to 30 bp in multiple rounds of detection, and can quickly and efficiently remove fluorescent detection probes for in situ sequencing; elution only requires incubation at 37°C and the elution solution is neutral, which will not damage the tissue and ensure that the tissue morphology is preserved intact; it will not introduce background, and can reduce the tissue background generated by multiple elutions, shortening the reaction time of the entire process.

[0007] In the related technology, the detection probe elution strategy has a long clearing time or a large number of clearing times, which will introduce a certain background, affect the subsequent rounds of fluorescence imaging, reduce the signal-to-noise ratio, and reduce the signal detection amount of the method. At the same time, the introduction of background limits the target throughput that can be detected by the in situ sequencing method.

[0008] In a first aspect, the present application provides an eluent for removing a fluorescent detection probe, using the following technical solution:

[0009] The invention discloses an eluting solution for removing a fluorescent detection probe, wherein the eluting solution is a 50-80% formamide solution containing 1×SSC, 5-10% SDS and 20-30mM Tris-HCl.

[0010] Optionally, the elution solution is a 50% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl.

[0011] Optionally, the elution solution can quickly remove the fluorescent detection probe for in situ sequencing.

[0012] Optionally, the treatment time of the eluent is 5 min.

[0013] Optionally, the eluent does not introduce background.

[0014] Optionally, the eluent is non-damaging to the tissue.

[0015] In a second aspect, the present application provides an elution method for removing a fluorescent detection probe, wherein the elution method utilizes the above-mentioned elution solution.

[0016] Optionally, the elution method is: immerse the glass slide in 1×PBS until the coverslip falls off, wash off the sealing medium, add the above elution solution, wash at 37°C for 5 minutes, and after the treatment is completed, confirm that there is no residual fluorescence under a fluorescence microscope before conducting the next round of detection.

[0017] In a third aspect, the present application provides an RNA in situ detection method, which adopts the following technical solution:

[0018] An RNA in situ detection method requires the introduction of a probe elution operation between each round of sequencing imaging.

[0019] In summary, the present application includes at least one of the following beneficial technical effects:

[0020] (1) The eluent provided in this application is mainly composed of a 50% formamide solution containing 1×SSC, 5% SDS, and 20mM Tris-HCl. When using the eluent, the reaction solution must be fully mixed in advance, the tissue is completely covered with the eluent, and incubated at 37°C for 5 minutes to effectively remove the fluorescent detection probe.

[0021] The technical solution provided in the present application is used to remove fluorescent detection probes with a length of 20 to 30 bp in multiple rounds of detection, and can quickly and efficiently remove fluorescent detection probes for in situ sequencing; elution only requires incubation at 37°C and the elution solution is neutral, which will not damage the tissue and ensure that the tissue morphology is preserved intact; it will not introduce background, and can reduce the tissue background generated by multiple elutions, shortening the reaction time of the entire process.

[0022] In the related technology, the detection probe elution strategy has a long clearing time or a large number of clearing times, which will introduce a certain background, affect the subsequent rounds of fluorescence imaging, reduce the signal-to-noise ratio, and reduce the signal detection amount of the method. At the same time, the introduction of background limits the target throughput that can be detected by the in situ sequencing method.

[0023] (2) Compared with the original 100% formamide eluent, the eluent of the present application has a higher signal-to-noise ratio after multiple rounds of washing, and the cell nucleus background of the tissue will not increase significantly.

[0024] (3) The elution solution of the present application can be used to quickly complete elution, and each elution only takes 5 minutes to completely remove the detection probe, which can reduce the overall detection time of the scheme.

[0025] The elution can be completed efficiently using the eluent of the present application, and the cleaning effect of a single elution is equivalent to or even better than that of multiple elutions using the original 100% formamide.

[0026] After elution with the elution solution of the present application, the tissue morphology remains intact and does not cause an increase in tissue background.

[0027] Since the most common way to increase target throughput is to increase the number of sequencing rounds, and the increase in background will limit the number of rounds that can be achieved by the technical route, the present application can effectively elute the fluorescent detection probe without introducing background, thereby increasing the target throughput that can be detected by this technical route.

[0028] By using the elution solution of the present application for elution, the signal can be effectively identified from the high background in the original scheme, thereby increasing the signal detection amount after multiple rounds of decoding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The microscopic imaging results of the first round using the eluent for the examples and comparative examples (the left side is the result of the examples, and the right side is the result of the comparative examples).

[0030] Figure 2 The fourth round of microscopic imaging results of the embodiment and the comparative example using the eluent are shown (the left side shows the results of the embodiment, and the right side shows the results of the comparative example).

[0031] The DAPI cell nucleus channel is not included in the figure, and the small dots of different colors are real signals. Figure 2 White solid shadows showing the comparative microscopic imaging results. DETAILED DESCRIPTION

[0032] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0034] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0035] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0036] The eluent provided in this application is mainly composed of a 50% formamide solution containing 1×SSC, 5% SDS, and 20mM Tris-HCl. When using the eluent, the reaction solution must be fully mixed in advance, the tissue is completely covered with the eluent, and incubated at 37°C for 5 minutes to effectively remove the fluorescent detection probe.

[0037] The technical solution provided in the present application is used to remove fluorescent detection probes with a length of 20 to 30 bp in multiple rounds of detection, and can quickly and efficiently remove fluorescent detection probes for in situ sequencing; elution only requires incubation at 37°C and the elution solution is neutral, which will not damage the tissue and ensure that the tissue morphology is preserved intact; it will not introduce background, and can reduce the tissue background generated by multiple elutions, shortening the reaction time of the entire process.

[0038] In the related technology, the detection probe elution strategy has a long clearing time or a large number of clearing times, which will introduce a certain background, affect the subsequent rounds of fluorescence imaging, reduce the signal-to-noise ratio, and reduce the signal detection amount of the method. At the same time, the introduction of background limits the target throughput that can be detected by the in situ sequencing method.

[0039] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0040] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] The present application is further described in detail below in conjunction with the embodiments, drawings and test results.

[0042] On breast cancer paraffin serial sections, the elution scheme of the present application (Example) and the elution scheme of the related technology (Comparative Example) were used to perform RNA in situ sequencing with 4 rounds of detection and 3 elutions to examine the difference in elution performance of the two schemes, that is, except for the difference in the elution schemes, other conditions were the same.

[0043] Example

[0044] Example 1

[0045] This embodiment provides an in-situ detection method.

[0046] The method specifically comprises the following steps:

[0047] 1. Tissue pretreatment of paraffin samples

[0048] (1) Baking: Bake the FFPE tissue sections in an oven at 60°C for 30 min.

[0049] (2) Dewaxing and rehydration: Immerse the sections in two cylinders of xylene at room temperature to ensure that the tissue is fully covered. Dewax for 15 min and 10 min respectively. Then immerse the sections in two cylinders of 100% alcohol for 2 min each. Wash the tissue three times with PBST (1× PBS containing 0.1% Tween-20).

[0050] (3) Fixation: Add 4% PFA (Biyuntian) to the tissue, discard the liquid after fixation for 10 minutes, and wash the tissue three times with PBST.

[0051] (4) Permeabilization: 0.1 M hydrochloric acid containing 0.3 mg / mL pepsin (Sigma) was added to the tissue and permeabilized at 37°C for 30 min. The tissue was then removed and washed three times with PBST.

[0052] (5) Dehydration and drying: Immerse the sections in 70%, 85% and 100% ethanol for gradient dehydration, each time for 1 min.

[0053] (6) Draw the hydrophobic circle: After the slices are dried, use Imm Edge TM Use a pen to draw a hydrophobic circle with a diameter of 1 cm around the tissue. After the hydrophobic circle is dry, wash the tissue inside the hydrophobic circle three times with PBST.

[0054] (II) In-situ testing process

[0055] (1) Dual-ligation probe hybridization

[0056] Add 50 μL of hybridization mixture containing 10 mM double-ligation probe (Shanghai Bioengineering, the same as double-ligation probe below), 6×SSC (Sigma), and 10% formamide (Sigma) to the tissue, and hybridize at 46°C for 4 h to allow the double-ligation probes designed for different target genes to fully bind to their targets, and then wash the tissue three times with PBST; then use a probe washing solution containing 2×SSC and 20% formamide to wash the tissue three times at 46°C for 5 min each time, and then wash the tissue three times with PBST.

[0057] The probe sequences used are shown in Table 1, specifically the nucleotide sequences shown in SEQ ID NO 1-260.

[0058] Table 1 Probe sequences used

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] (2) Double-ligation probe ring formation

[0067] 1) First step of ligation of dual ligation probe

[0068] 50 μL of a ligation reaction mixture containing 25% glycerol (Sigma), 0.2 μg / μL BSA, 1× SplintR buffer (NEB), 0.25 U / μL SplintR ligase (NEB), and 1 U / μL RiboLock RNase inhibitor (Thermo) was added to the tissue and incubated at 37°C for 30 min, followed by washing the tissue three times with PBST.

[0069] 2) Double-ligation probe circularization

[0070] 50 μL of a looping mixture containing 0.5 μM looping splint ST, 2 mM ATP, 0.2 μg / μL BSA, 1xT4 DNA Ligase buffer, 1 U / μL T4 DNA Ligase and 1 U / μL RiboLock RNase inhibitor was added to the tissue and incubated at 37°C for 30 min, followed by washing the tissue three times with PBST. The looping splint sequence is the nucleotide sequence shown in SEQ ID NO: 261, specifically: GGCTCCACTAAATAGACGCA.

[0071] (3) Rolling circle amplification

[0072] Add 50 μL of rolling circle amplification reaction solution containing 1 mM dNTP (Thermo), 5% glycerol, 0.2 μg / μL BSA, 1×phi29 buffer, 1 U / μL phi29 DNA polymerase and 1 U / μL RiboLock RNase inhibitor to the tissue, incubate at 30°C for 12-16 hours, and then wash the tissue three times with PBST.

[0073] (4) Four-wheel probe detection and imaging

[0074] Add 50 μL of sequencing reaction mixture containing 0.2 μM anchor primer, 0.1 μM of each fluorescent detection probe, 1×T4 DNA Ligase buffer, 1 mM ATP and 0.1 U / μL T4 DNA Ligase to the tissue and incubate at 30°C for 45 min; then wash the tissue three times with PBST. After the tissue is naturally air-dried, add Slowfade anti-fluorescence quencher (Thermo) containing 0.5 μg / mL DAPI and incubate at room temperature for about 5 min before fluorescence microscopy imaging.

[0075] The fluorescence detection probe sequences are shown in Table 2. Specifically, they are nucleotide sequences shown in SEQ ID NOs: 262-279.

[0076] Table 2 Fluorescence detection probe sequences

[0077]

[0078] (6) Detection probe elution

[0079] Probe elution operation needs to be introduced between each round of sequencing imaging. First, the glass slide is placed in 1×PBS and soaked until the coverslip falls off. After washing away the sealing medium, add 50% formamide solution containing 1×SSC, 5% SDS, and 20mM Tris-HCl, and wash at 37°C for 5 minutes. After the treatment is completed and no fluorescence is confirmed under a fluorescence microscope, the next round of detection can be carried out. This cycle completes 4 rounds of sequencing imaging.

[0080] Example 2

[0081] This embodiment provides an in-situ detection method.

[0082] The difference between this embodiment and embodiment 1 is that: (6) the elution solution used in eluting the detection probe is a 50% formamide solution containing 1×SSC, 10% SDS, and 20 mM Tris-HCl. The steps are consistent with those in embodiment 1.

[0083] Example 3

[0084] This embodiment provides an in-situ detection method.

[0085] The difference between this embodiment and embodiment 1 is that: (6) the elution solution used in eluting the detection probe is a 50% formamide solution containing 1×SSC, 5% SDS, and 25 mM Tris-HCl. The steps are consistent with those in embodiment 1.

[0086] Example 4

[0087] This embodiment provides an in-situ detection method.

[0088] The difference between this embodiment and embodiment 1 is that: (6) the elution solution used in eluting the detection probe is a 50% formamide solution containing 1×SSC, 5% SDS, and 30 mM Tris-HCl. The steps are consistent with those in embodiment 1.

[0089] Example 5

[0090] This embodiment provides an in-situ detection method.

[0091] The difference between this embodiment and embodiment 1 is that: (6) the elution solution used in eluting the detection probe is a 60% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl. The steps are consistent with those in embodiment 1.

[0092] Example 6

[0093] This embodiment provides an in-situ detection method.

[0094] The difference between this embodiment and embodiment 1 is that: (6) the elution solution used in eluting the detection probe is an 80% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl. The steps are consistent with those in embodiment 1.

[0095] Comparative Example

[0096] Comparative Example 1

[0097] This comparative example provides an in-situ detection method.

[0098] The difference between this comparative example and Example 1 is that in the step (6) of eluting the detection probe, after washing away the sealing agent, 100% formamide was added and washed at 37°C for 3 times, each time for 10 minutes. After the treatment was completed, it was confirmed under a fluorescence microscope that there was no residual fluorescence before the next round of detection was performed, and 4 rounds of sequencing imaging were completed in this cycle. The rest of the operation steps were consistent with those of the example.

[0099] Comparative Example 2

[0100] This comparative example provides an in-situ detection method.

[0101] The difference between this comparative example and Example 1 is that: (6) the elution solution used in eluting the detection probe is a 50% formamide solution containing 1×SSC, 5% SDS, and 15 mM Tris-HCl. The steps are consistent with those in Example 1.

[0102] Comparative Example 3

[0103] This comparative example provides an in-situ detection method.

[0104] The difference between this comparative example and Example 1 is that: (6) the elution solution used in eluting the detection probe is a 50% formamide solution containing 1×SSC, 5% SDS, and 35 mM Tris-HCl. The steps are consistent with those in Example 1.

[0105] Comparative Example 4

[0106] This comparative example provides an in-situ detection method.

[0107] The difference between this comparative example and Example 1 is that: (6) the elution solution used in eluting the detection probe is a 40% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl. The steps are consistent with those in Example 1.

[0108] Comparative Example 5

[0109] This comparative example provides an in-situ detection method.

[0110] The difference between this comparative example and Example 1 is that: (6) the elution solution used in eluting the detection probe is a 90% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl. The steps are consistent with those in Example 1.

[0111] Test results

[0112] Compare the test results of Examples 1-6 and Comparative Examples 1-5. Figure 1-2 shown.

[0113] in, Figure 1 The first round of microscopic imaging results using the eluent, the left side is the result of the embodiment, and the right side is the result of the comparative example; Figure 2 The fourth round of microscopic imaging results using the eluent are shown in FIG. 1 , wherein the left side shows the results of the embodiment, and the right side shows the results of the comparative example.

[0114] Depend on Figure 1 and Figure 2 It can be seen that the two fluorescent detection probe elution schemes of Example 1 and Comparative Example 1 have no obvious signal residue after each round of cleaning, indicating that the technical solution (embodiment) of the present application can achieve a probe removal effect equivalent to that of the related technical solution (comparative example). And after 3 rounds of elution, the related technical solution (comparative example) has a serious tissue background, while the technical solution (embodiment) of the present application has no obvious background, indicating that the technical solution of the present application can be applied to the probe elution of more rounds of sequencing, and will not introduce obvious background.

[0115] Meanwhile, it can be seen from the test that the results of Example 2-6 are almost indistinguishable from the imaging results of Example 1, and no obvious background appears, while the results of Comparative Example 2-5 show the presence of visible tissue background.

[0116] The cleaning scheme of the present application can complete multiple rounds of washing within 5 minutes, and will not cause an increase in tissue background in subsequent rounds such as the fourth round; while the cleaning scheme in the related art requires 100% formamide to be washed three times at 37°C, each time for 10 minutes to clean the fluorescence signal of the previous round, and subsequent rounds will introduce serious tissue background.

[0117] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An eluent for removing a fluorescent detection probe, characterized in that: The elution solution is a 50-80% formamide solution containing 1×SSC, 5-10% SDS, and 20-30 mM Tris-HCl.

2. The eluent according to claim 1, characterized in that The eluent is a 50% formamide solution containing 1×SSC, 5% SDS, and 20 mM Tris-HCl.

3. The eluent according to claim 1, characterized in that The eluent can quickly remove the fluorescent detection probe for in situ sequencing.

4. The eluent according to claim 1, characterized in that The treatment time of the eluent is 5 min.

5. The eluent according to claim 1, characterized in that When using, the tissue should be completely covered with the washing solution.

6. The eluent according to claim 1, characterized in that The eluent does not introduce background.

7. The eluent according to claim 1, characterized in that The eluent does not damage tissue.

8. A method for removing a fluorescent detection probe, characterized in that: The elution method utilizes the elution solution according to any one of claims 1 to 7.

9. The elution method according to claim 8, characterized in that The elution method is as follows: immerse the glass slide in 1×PBS until the cover glass falls off, wash off the sealing medium, add the elution solution described in any one of claims 1 to 7, wash at 37°C for 5 minutes, and after the treatment is completed, confirm that there is no residual fluorescence under a fluorescence microscope before conducting the next round of detection.

10. A method for in situ detection of RNA, characterized in that: The RNA in situ detection method requires the introduction of a probe elution operation between each round of sequencing imaging.

Images