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In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs
miRNA的原位杂交(ISH)和量子点(QD)   

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Abstract

miRNA are short non-coding RNA which inhibit translation of mRNA. miRNA regulate several cellular processes. Certain miRNA are known to induce oncogenesis. miRNA can be measured by real-time PCR and be imaged using a combination of in situ hybridization (ISH) and quantum dots (QD). The advantage of using quantum dots is that several miRNA can be simultaneously measured using multiplexed QD. Additionally, miRNA can be visualized in different regions of the tissue. Since miRNA are biomarkers of various disease states, miRNA can be visualized and quantitated in tissue sections for diagnostic and prognostic purposes. Here we describe ISH-QD analysis of tissue sections. Tissue sections from xenografts or clinical specimens are used. These are deparaffinized, treated with Proteinase K and hybridized with a biotin-probe to specific to the miRNA. The in situ hybridization is performed by labeling the biotin-probes and followed by labeling with streptavidin tagged quantum dots. Image acquisition of the quantum dots is performed and analyzed for the miRNA expression levels. Combining ISH and QD gives a powerful tool to detect miRNA in different cells of the tissue.

Keywords: miRNA(miRNA), in situ hybridization(原位杂交), Multiplexed quantum dots(多路复用量子点), Cancer(癌症), Cancer associated stroma(癌症相关基质), Cellular compartments(细胞区室), Biomarkers(生物标志物), Tissue staining(组织染色)

Background

miRNAs can be easily detected by quantitative real-time PCR or Northern blotting. However, imaging miRNAs has been challenging. Recent advances in quantum dots imaging have made it possible to determine expression of miRNAs in tissues. Using this process, miRNAs can be visualized in different compartments of a tissue, such as tumor, stroma, immune cells, etc. Additionally, miRNAs can be multiplexed to determine co-localization of miRNAs which mediate specific processes, in different cellular regions. Different tissues can be used for ISH-QD such as tissues from xenografts or human clinical samples. Tissues from animal studies are formalin fixed and paraffin embedded. These tissues were used for ISH-QD analysis (Figure 1).


Figure 1. In situ hybridization coupled to Quantum dots labelling for visualization of miRNA. In the ISH-QD protocol, miRNA are detected on formalin fixed paraffin embedded tissue sections using biotinylated miRNA probes binding to streptavidin-conjugated QDs. The specific QD gives a specific fluorescent signal which is quantified using the Inform v1.3 software.

Materials and Reagents

  1. Gloves
  2. Tissue slides
  3. Superfrost plus slides
  4. Coverslips
  5. LNATM Scramble-miR probe (Exiqon, catalog number: 699004-370 )
  6. LNATM miR-409-3p 5’ biotin labeled (Exiqon, catalog number: 610701-370 )
  7. LNATM miR-409-5p 5’ biotin labeled (Exiqon, catalog number: 615615-370 )
  8. RNaseZap (Thermo Fisher Scientific, AmbionTM)
  9. Xylene
  10. Ethanol
  11. Sterile PBS (pH 7)
  12. Nail polish
  13. Horse serum (Vector Laboratories, catalog number: S-2000 )
  14. Streptavidin block reagent (Thermo Fisher Scientific, InvitrogenTM)
  15. Streptavidin conjugated QDs 625 nm (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A10196 ) (1 µm stock from Invitrogen)
  16. Streptavidin conjugated QDs 565 nm (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: Q10131MP ) (1 µm stock from Invitrogen)
  17. 6% IgG-free, protease free BSA (Jackson ImmunoResearch, catalog number: 001-000-162 )
  18. BSA
  19. 0.4% Triton X-100
  20. 0.1% Tween-20
  21. Mounting media
  22. 4’6-diamidino-2-phenylindole (DAPI) (Vector Laboratories)
  23. 1 M Tris-HCl (pH 7.4)
  24. 0.5 M EDTA
  25. NaCl
  26. RNase free Milli-Q water, autoclaved (all solutions prepared in this water)
  27. Exiqon microRNA ISH buffer set and Proteinase K (Exiqon, catalog number: 90000 )
  28. 20x SSC, pH 7.0
  29. Proteinase K stock (see Recipes)
  30. Proteinase K buffer (see Recipes)
  31. Proteinase K solution (15 µg/ml) (see Recipes)
  32. SSC solutions (see Recipes)
  33. Hybridization mix (see Recipes)
  34. Streptavidin blocking solution (see Recipes)
  35. QD solutions (see Recipes)
  36. PBS-T (see Recipes)

Equipment

  1. Autoclave
  2. Hybridizer (DAKO Statspin Hybridizer)
  3. Glass cutter
  4. Slide rack and glass jars
  5. PAP pen or ImmEdge pen (Vector Laboratories, catalog number: H-4000 )
  6. Water bath
  7. CRi multi-spectral camera with built-in Nuance software and inForm software (PerkinElmer, Waltham, MA)

Software

  1. Nuance v3.1 software
  2. Inform v1.3 software
  3. Graphpad Prism software

Procedure

Notes:

  1. The in situ hybridization procedure has been described in detail in the Exiqon ISH protocol.
  2. Caution of RNA work
    1. All steps should take place in a clean and nuclease free environment.
    2. All surfaces should be cleaned with RNaseZap.
    3. Wear gloves at all times and use autoclaved glassware and RNase grade MilliQ water which is autoclaved for preparation of all solutions.
    4. In situ hybridization was performed as mentioned in the Exiqon manual.
  3. Optimization
    1. Optimization of Proteinase K treatment required before the start of the actual assay. Proteinase K reagent prepared immediately before use at a concentration of 15 µg/ml.
    2. Optimization of LNA miRNA probe and scramble miRNA. A probe concentration of 80 nM was optimal for detection of miR-409-3p and miR-409-5p.
  4. Reagents to be prepared on the day of the experiment
    1. Proteinase K
    2. Hybridization mix: Place the appropriate amount of probe in a 2 ml non-stick RNase-free tube. Denature the probe at 90 °C for 4 min. Centrifuge the tubes and spin down. Add 2 ml of 1x miRNA ISH buffer.
  1. Deparaffinize slides in xylene and ethanol
    1. Xylene - 5 min, 3 times in a glass jar
    2. 99.9% ethanol - 10 min, 2 times, in a glass jar
    3. 99.9% ethanol - 5 min, 1 time, in a glass jar
    4. 96% ethanol - 10 min, 1 time, in a glass jar
    5. 96% ethanol - 5 min, 1 time, in a glass jar
    6. 70% ethanol - 10 min, 1 time, in a glass jar
    7. 70% ethanol - 5 min, 1 time, in a glass jar
    8. PBS - 2-5 min, each 1 time, in a glass jar

  2. Proteinase K treatment and wash
    1. Add Proteinase K to Proteinase K buffer (final concentration 15 µg/ml) (add 7.5 µl Proteinase K stock to 10 ml Proteinase K buffer). 
    2. Add sufficient volume of proteinase K solution to cover the slides (instructions in the Exiqon manual).
    3. Incubate for 10 min, at 37 °C, in a Hybridizer.
    4. Transfer the slides into a new glass jar containing PBS. Repeat this step twice.

  3. Hybridization
    1. Use PAP pen to mark around the tissue section.
    2. Using a glass cutter cut the coverslip (autoclaved) to the size of the tumor tissue section.
    3. Add miRNA detection probe (50 µl, hybridization mix) on the tissue section. Avoid air bubbles.
    4. If a slide has several serial tissues sections of the same tumor, it can be used to determine several probes (such as miR-409-3p, miR-409-5p and Scramble miRNA). Once the probe is added, place the cut coverslip very carefully without trapping air bubbles.
    5. Seal the edge of the coverslip to the slide using colorless nail polish. Let it dry.
    6. Place the slides in the Hybridizer at 55 °C for 1 h.

  4. Disassembly and washes
    1. Carefully remove the coverslips.
    2. Place the slides into a glass jar with 5x SSC in a water bath at 55 °C.
      Note: Use a water bath at 55 °C to do the washes in glass jars. The graded SSC solutions were prepared with autoclaved water.
    3. 5x SSC - 5 min 55 °C
    4. 1x SSC - 5 min 55 °C
    5. 1x SSC - 5 min 55 °C
    6. 0.2x SSC - 5 min 55 °C
    7. 0.2x SSC - 5 min 55 °C
    8. 0.2x SSC - 5 min RT
    9. Incubate the slides in PBS.

  5. Blocking
    The slides were incubated in PBS containing 2.5% horse serum and 20% streptavidin block reagent for 1 h at room temperature.

  6. Single QD labeling
    1. Streptavidin-conjugated QDs (565 nm [green] and 625 nm [red]) were prepared at a 10 nM concertation in PBS-T with 6% IgG-free, protease free BSA.
    2. The slides were incubated at 37 °C for 60 min.

  7. Washing
    The slides were washed 3 times for 5 min each in PBS-T (PBS + 0.4% Triton X-100 + 0.1% Tween-20).

  8. Mounting
    Cover the slides with cover glass using aqueous mounting media containing 4’6-diamidino-2-phenylindole (DAPI).
     
  9. Image acquisition
    The CRi spectral imaging system was used. Multispectral images were taken using the Nuance v3.1 software. For each field of the tissue, serial images were taken from 450 to 800 nm with an interval of 10 nm. This generates a cube a stack of 36 separate images at different wavelengths and has spectral information for every pixel in the image. Images are acquired at 400x magnification (Figure 2).


    Figure 2. Representative images of multiplexed image of miR-409-3p (green) and miR-409-5p (red) on a human prostate cancer tissue sample of Gleason grade 8, detected using in situ hybridization and quantum dots labeling. DAPI was stained in blue. Scrambled miRNA was used as a negative control, on a adjacent section on an separate slide of the same tissue. Hematoxylin and eosin staining (H&E) was also performed. (Magnification 40x) 

Data analysis

Once data acquisition is performed the images are deconvoluted or unmixed to extract the specific signal by a specified QD. A spectral library was built for 565 and 625 nm. The spectral library was used to unmix the cube. Autofluorescence was reduced using the Real Component Analysis plug-in software. The images after unmixing represented the distribution of QDs on the tissue. Signal was quantified using Inform v1.3 software. The software discriminates cancer and stromal (non-cancer) areas. The software also discriminates between nuclear and cytoplasmic area based on the DAPI stain. Using this software the QD signal from the tumor and stromal sections can be determined and quantified. Numerical values are generated for the image and the cytoplasmic component of the tumor and stromal regions is used for plotting the data. The data is graphically plotted using Graphpad Prism software. Data distribution was depicted in box plot formats. Statistical analysis of tissue array was performed using a non-parametric Wilcoxon rank sum test. Values of P < 0.05 were considered to be statistically significant.

Notes

  1. Multiplexed QD
    1. Principle: Multiplexed ISH-QD labeling involves using a single tissue which is labeled with a miRNA-biotin probe using ISH and followed by a streptavidin blocking step and incubated with streptavidin conjugated-QD. This process is repeated with another miRNA-biotin probe and another (different wavelength) streptavidin conjugated-QD pair. Finally the tissue is stained with mounting media containing DAPI. Thus, an image is generated for different miRNA with different active fluorescent QD on a single tissue section.
    2. Procedure: Slides were stained sequentially, with one miRNA probe and a QD procedure followed by another probe and QD. After the first QD labeling, slides were washed 3 times for 5 min each with PBS-T. Slides were then used for hybridization (Procedure C) with another miRNA probe and continued to Procedure H. Hybridization mix and probes stable for only 6 months, avoid multiple freeze-thaw cycles.
  2. Freshly prepared tissue slides give better miRNA signals. Older tissue preparations, may have loss of miRNA content due to RNase degradation over time.

Recipes

  1. Proteinase K stock
    Reconstitute 20 mg/ml in 600 µl of 10 mM Tris-HCl, pH 7.4 (RNase-free). Store at -20 °C
  2. Proteinase K buffer
    5 ml of 1 M Tris-HCl (pH 7.4)
    2 ml of 0.5 M EDTA
    0.2 ml of 5 M NaCl
    900 ml of RNase-free water
    Adjust the volume to 1,000 ml and autoclave
  3. Proteinase K solution (15 µg/ml)
    Before the start of the experiment add 7.5 µl of Proteinase K stock to 10 ml Proteinase K buffer to make a concentration of 15 µg/ml
  4. SSC solutions
    Stock: 20x SSC solution
    5x SSC: 1 L = 250 ml 20x SSC + 750 ml water
    5x SSC: 1 L = 50 ml 20x SSC + 950 ml water
    5x SSC: 1 L = 10 ml 20x SSC + 990 ml water
    All solutions to be autoclaved
  5. Hybridization mix (miRNA ISH buffer and LNATM detection probes)
    Note: LNATM miRNA detection probes come in 40 µl volume at a 25 µM concentration.
    1. Add 1 ml 2x ISH buffer to 1 ml of RNase free water
    2. Denature the LNATM miRNA detection probes at 90 °C for 4 min before adding the ISH buffer. Spin down the solution
    3. For making 80 nM of miRNA probes, add 6.4 µl of denatured probe (25 µM) to 2 ml of 1x ISH buffer
      Note: Hybridization mix can be stored at -20 °C for six months.
  6. Streptavidin blocking solution
    PBS containing 2.5% horse serum and 20% streptavidin block reagent
  7. QD solutions
    Streptavidin-conjugated QDs (565 nm [green] and 625 nm [red]) was prepared at a 10 nM concertation in PBS with 6% IgG-free, protease free BSA
  8. PBS-T (PBS + 0.4% Triton X-100 + 0.1% Tween-20)
    Add 1 ml of Tween-20 and 4 ml of Triton X-100 to 1 L PBS
    Autoclave at 121 °C for 15 min

Acknowledgments

This procedure has been published in part in the paper by Hu et al., 2011 and Josson et al., 2015. The in situ hybridization procedure has been optimized from the Exiqon ISH protocol. Grant support for this work is from P01-CA98912, DAMD-17-03-02-0033, RO1-CA122602 (L.W.K. Chung).

References

  1. Hu, P., Chu, G. C., Zhu, G., Yang, H., Luthringer, D., Prins, G., Habib, F., Wang, Y., Wang, R., Chung, L. W. and Zhau, H. E. (2011). Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer. PLoS One 6(12): e28670.
  2. Josson, S., Gururajan, M., Sung, S. Y., Hu, P., Shao, C., Zhau, H. E., Liu, C., Lichterman, J., Duan, P., Li, Q., Rogatko, A., Posadas, E. M., Haga, C. L. and Chung, L. W. (2015). Stromal fibroblast-derived miR-409 promotes epithelial-to-mesenchymal transition and prostate tumorigenesis. Oncogene 34(21): 2690-2699.

简介

miRNA是抑制mRNA翻译的短非编码RNA。 miRNA调节几种细胞过程。已知某些miRNA诱导肿瘤发生。 miRNA可以通过实时PCR测量,并使用原位杂交(ISH)和量子点(QD)的组合进行成像。使用量子点的优点是可以使用多重QD同时测量几种miRNA。此外,miRNA可以在组织的不同区域可视化。由于miRNA是各种疾病状态的生物标志物,miRNA可以在组织切片中进行可视化和定量,以用于诊断和预后。这里我们描述组织切片的ISH-QD分析。使用来自异种移植物或临床标本的组织切片。将其脱石蜡,用蛋白酶K处理并与生物素探针杂交以对miRNA具有特异性。原位杂交通过标记生物素探针进行,然后用链霉亲和素标记的量子点进行标记。进行量子点的图像采集并分析其miRNA表达水平。结合ISH和QD为检测组织不同细胞中的miRNA提供了强大的工具。

背景 通过定量实时PCR或Northern印迹可以很容易地检测miRNA。然而,成像miRNA一直是挑战性的。量子点成像的最新进展使得可以确定组织中miRNAs的表达。使用这个过程,miRNA可以在组织的不同隔室中显现,例如肿瘤,基质,免疫细胞等。另外,miRNA可以被多重化以确定介导特定过程的miRNA的共定位,在不同的细胞区域。不同的组织可用于ISH-QD,例如来自异种移植物或人类临床样品的组织。来自动物研究的组织是福尔马林固定和石蜡包埋的。这些组织用于ISH-QD分析(图1)。


在ISH-QD方案中,使用生物转化的miRNA在福尔马林固定石蜡包埋的组织切片上检测miRNA探针结合链霉抗生物素蛋白偶联的量子点。具体的QD给出了使用Inform v1.3软件量化的特定荧光信号。

关键字:miRNA, 原位杂交, 多路复用量子点, 癌症, 癌症相关基质, 细胞区室, 生物标志物, 组织染色

材料和试剂

  1. 手套
  2. 组织幻灯片
  3. 超级霜加幻灯片
  4. 盖帽
  5. LNA TM Scramble-miR探针(Exiqon,目录号:699004-370)
  6. LNA TM miR-409-3p 5'生物素标记(Exiqon,目录号:610701-370)
  7. LNA TM 标记的miR-409-5p 5'生物素(Exiqon,目录号:615615-370)
  8. RNaseZap(Thermo Fisher Scientific,Ambion TM
  9. 二甲苯
  10. 乙醇
  11. 无菌PBS(pH 7)
  12. 指甲油
  13. 马血清(Vector Laboratories,目录号:S-2000)
  14. 链霉亲和素嵌段试剂(Thermo Fisher Scientific,Invitrogen TM
  15. 链霉亲和素偶联的QD 625nm(Thermo Fisher Scientific,Molecular Probes TM,目录号:A10196)(来自Invitrogen的1μm存货)
  16. 链霉亲和素缀合的QD 565nm(Thermo Fisher Scientific,Molecular Probes TM,目录号:Q10131MP)(来自Invitrogen的1μm存货)
  17. 6%无IgG,无蛋白酶BSA(Jackson ImmunoResearch,目录号:001-000-162)
  18. BSA
  19. 0.4%Triton X-100
  20. 0.1%Tween-20
  21. 安装媒体
  22. 4'-二脒基-2-苯基吲哚(DAPI)(Vector Laboratories)
  23. 1M Tris-HCl(pH7.4)
  24. 0.5 M EDTA
  25. NaCl
  26. 无RNase的Milli-Q水,高压灭菌(在该水中制备的所有溶液)
  27. Exiqon microRNA ISH缓冲液组和蛋白酶K(Exiqon,目录号:90000)
  28. 20x SSC,pH 7.0
  29. 蛋白酶K库存(见食谱)
  30. 蛋白酶K缓冲液(参见食谱)
  31. 蛋白酶K溶液(15μg/ml)(参见食谱)
  32. SSC解决方案(见配方)
  33. 杂交组合(见配方)
  34. 链霉亲和素阻断溶液(参见食谱)
  35. QD解决方案(见配方)
  36. PBS-T(见食谱)

设备

  1. 高压灭菌器
  2. 混合器(DAKO Statspin Hybridizer)
  3. 玻璃切割机
  4. 滑架和玻璃瓶
  5. PAP笔或ImmEdge笔(Vector Laboratories,目录号:H-4000)
  6. 水浴
  7. CRi多光谱摄像机,内置Nuance软件和InForm软件(PerkinElmer,Waltham,MA)

软件

  1. Nuance v3.1软件
  2. 通知v1.3软件
  3. Graphpad Prism软件

程序

注意:

  1. 原位杂交程序已在Exiqon ISH协议中详细描述。
  2. 注意RNA工作
    1. 所有步骤都应该在干净,无核酸的环境中进行。
    2. 所有表面均应用RNaseZap清洁。
    3. 请始终佩戴手套,并使用高压灭菌的玻璃器皿和RNase级MilliQ水进行高压灭菌以制备所有溶液。
    4. 如Exiqon手册中所述进行原位杂交。
  3. 优化
    1. 在实际测定开始之前需要优化蛋白酶K处理。蛋白酶K试剂在使用前立即以15μg/ml的浓度制备。
    2. LNA miRNA探针和加扰miRNA的优化。 80nM的探针浓度对于检测miR-409-3p和miR-409-5p是最佳的
  4. 在实验当天准备的试剂
    1. 蛋白酶K
    2. 杂交混合物:将适量的探针置于2ml不粘核糖核酸酶管中。探头在90℃变形4分钟。离心管并旋转下来。加入2毫升1x miRNA ISH缓冲液
  1. 在二甲苯和乙醇中脱蜡
    1. 二甲苯5分钟,玻璃瓶中3次
    2. 99.9%乙醇 - 10分钟,2次,在玻璃瓶中
    3. 99.9%乙醇 - 5分钟,1次,在玻璃瓶中
    4. 96%乙醇-10分钟,1次,在玻璃瓶中
    5. 96%乙醇 - 5分钟,1次,在玻璃瓶中
    6. 70%乙醇 - 10分钟,1次,在玻璃瓶子里
    7. 70%乙醇 - 5分钟,1次,在玻璃瓶子里
    8. PBS - 2-5分钟,每次1次,在玻璃瓶中

  2. 蛋白酶K处理和洗涤
    1. 将蛋白酶K加入蛋白酶K缓冲液(终浓度15μg/ml)(加入7.5μl蛋白酶K储存液至10ml蛋白酶K缓冲液)。 
    2. 加入足够体积的蛋白酶K溶液以覆盖幻灯片(Exiqon手册中的说明)。
    3. 孵化10分钟,在37℃,在杂交器中。
    4. 将载玻片转移到含有PBS的新玻璃瓶中。重复此步骤。

  3. 杂交
    1. 使用PAP笔在组织部分周围标记。
    2. 使用玻璃切割机切割盖玻片(高压灭菌)至肿瘤组织切片的大小。
    3. 在组织切片上加入miRNA检测探针(50μl,杂交混合物)。避免气泡。
    4. 如果载玻片具有相同肿瘤的几个连续组织切片,则可用于确定几个探针(如miR-409-3p,miR-409-5p和Scramble miRNA)。一旦添加探头,请非常小心地放置切割的盖玻片,而不会吸入气泡。
    5. 使用无色指甲油将盖玻片的边缘密封到幻灯片。让它干
    6. 将幻灯片放置在55℃的Hybridizer中1小时。

  4. 拆卸和清洗
    1. 小心地取出盖玻片。
    2. 将载玻片放入带有5x SSC的玻璃瓶中,在55°C的水浴中 注意:使用55°C的水浴在玻璃瓶中进行洗涤。用高压灭菌水制备分级SSC溶液。
    3. 5x SSC - 5分55℃
    4. 1x SSC - 5分55℃
    5. 1x SSC - 5分55℃
    6. 0.2x SSC - 5分55℃
    7. 0.2x SSC - 5分55℃
    8. 0.2x SSC - 5分钟RT
    9. 在PBS中孵育幻灯片。

  5. 阻止
    将载玻片在含有2.5%马血清和20%链霉亲和素阻滞剂的PBS中在室温下孵育1小时。

  6. 单一QD标签
    1. 在含有6%无IgG的无蛋白酶BSA的PBS-T中以10nM共振制备链霉亲和素偶联的量子点(565nm [绿色]和625nm [红色])。
    2. 将载玻片在37℃下孵育60分钟
  7. 洗涤
    将载玻片在PBS-T(PBS + 0.4%Triton X-100 + 0.1%Tween-20)中洗涤3次,每次5分钟。

  8. 安装
    用含有4'6-二脒基-2-苯基吲哚(DAPI)的水性载体介质用盖玻片覆盖载玻片。
     
  9. 图像采集
    使用CRi光谱成像系统。使用Nuance v3.1软件拍摄多光谱图像。对于组织的每个区域,以450nm至800nm的间隔以10nm的间隔拍摄串行图像。这产生一个立方体,具有不同波长的36个不同图像的堆叠,并且具有图像中每个像素的光谱信息。以400倍放大倍率获取图像(图2)。


    图2.使用原位检测的Gleason等级8的人类前列腺癌组织样品上miR-409-3p(绿色)和miR-409-5p(红色)的多路复用图像的代表性图像,杂交和量子点标记。 DAPI染色为蓝色。加扰miRNA用作阴性对照,在相同组织的单独载玻片上的相邻区段上。苏木精和曙红染色(H& E)也得到了证实。 (放大40倍) 

数据分析

一旦执行了数据采集,图像被去卷积或不混合,以通过指定的QD提取特定信号。为565和625 nm建立了一个光谱库。使用光谱库来混合立方体。使用Real Component Analysis插件软件降低自动荧光。解混后的图像表示组织上QD的分布。使用Inform v1.3软件量化信号。该软件区分癌症和基质(非癌症)区域。该软件还基于DAPI染色来区分核和细胞质区域。使用该软件可以确定和定量来自肿瘤和基质切片的QD信号。产生图像的数值,肿瘤的细胞质成分和基质区用于绘制数据。数据使用Graphpad Prism软件进行图形化绘制。数据分布以箱形图形式进行了描述。使用非参数Wilcoxon秩和检验进行组织阵列的统计分析。 P 的值0.05被认为具有统计学意义。

笔记

  1. 多路复用QD
    1. 原理:多重ISH-QD标记涉及使用ISH标记的miRNA-生物素探针的单个组织,随后进行链霉抗生物素蛋白封闭步骤,并与链霉抗生物素蛋白缀合的QD一起温育。该过程与另一种miRNA-生物素探针和另一种(不同波长)的链霉亲和素偶联的QD对重复。最后,组织用含有DAPI的载体介质染色。因此,在单个组织切片上生成具有不同活性荧光QD的不同miRNA的图像
    2. 程序:顺序染色幻灯片,用一个miRNA探针和QD程序,然后是另一个探针和QD。在第一次QD标记后,将载玻片用PBS-T洗涤3次,每次5分钟。然后将幻灯片用于与另一种miRNA探针杂交(方法C),并继续进行步骤H.杂交混合物和探针稳定仅6个月,避免多次冻融循环。
  2. 新鲜制备的组织载玻片提供更好的miRNA信号。较老的组织制剂,由于RNase随时间的退化而可能会损失miRNA含量

食谱

  1. 蛋白酶K库存
    在600μl10 mM Tris-HCl,pH 7.4(不含RNase)中重构20 mg/ml。储存于-20°C
  2. 蛋白酶K缓冲液
    5ml 1M Tris-HCl(pH7.4)
    2 ml 0.5 M EDTA 0.2 ml 5 M NaCl 900毫升无RNase的水
    调节体积至1000毫升,高压釜
  3. 蛋白酶K溶液(15μg/ml)
    在实验开始之前,将7.5μl蛋白酶K储备液添加到10ml蛋白酶K缓冲液中以使浓度为15μg/ml
  4. SSC解决方案
    库存:20x SSC解决方案
    5×SSC:1L = 250ml 20×SSC + 750ml水 5×SSC:1L = 50ml 20×SSC + 950ml水
    5×SSC:1L = 10ml 20×SSC + 990ml水
    所有解决方案要高压灭菌
  5. 杂交组合(miRNA ISH缓冲液和LNA TM检测探针)
    注意:LNA TM miRNA检测探针的浓度为25μM,浓度为40μl。
    1. 将1ml 2x ISH缓冲液加入1ml无RNase的水中
    2. 在加入ISH缓冲液之前,在90℃下将LNA TM miRNA检测探针变性4分钟。旋转解决方案
    3. 为了制备80 nM的miRNA探针,加入6.4μl变性探针(25μM)至2 ml的1x ISH缓冲液, 注意:杂交组合可以在-20°C储存六个月。
  6. 链霉亲和素阻断溶液
    含有2.5%马血清和20%链霉抗生物素蛋白阻断剂的PBS/PBS
  7. QD解决方案
    在具有6%IgG,无蛋白酶BSA的PBS中以10nM共振制备链霉亲和素偶合的量子点(565nm [绿色]和625nm [红色]),
  8. PBS-T(PBS + 0.4%Triton X-100 + 0.1%吐温-20)
    加入1ml吐温-20和4ml Triton X-100至1μLPBS 高压灭菌在121°C 15分钟

Achnowledgments

该程序已部分公布于Hu等人,2011年和Josson等人,2015年。原位杂交程序已从Exiqon ISH协议进行优化。这项工作的授权来自P01-CA98912,DAMD-17-03-02-0033,RO1-CA122602(L.W.K. Chung)。

参考文献

  1. Hu,P.,Chu,GC,Zhu,G.,Yang,H.,Luthringer,D.,Prins,G.,Habib,F.,Wang,Y.,Wang,R.,Chung,LW和Zhau, HE(2011)。激活的c的多重量子点标签 - 在去势抗性人类前列腺癌中的信号传导。 6(12):e28670。
  2. Josson,S.,Gururajan,M.,Sung,SY,Hu,P.,Shao,C.,Zhau,HE,Liu,C.,Lichterman,J.,Duan,P.,Li,Q.,Rogatko, A.,Posadas,EM,Haga,CL和Chung,LW(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25065597"基质成纤维细胞衍生的miR-409促进上皮 - 间质转化和前列腺肿瘤发生。 34(21):2690-2699。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Josson, S., Gururajan, M. and Chung, L. W. (2017). In situ Hybridization (ISH) and Quantum Dots (QD) of miRNAs. Bio-protocol 7(4): e2138. DOI: 10.21769/BioProtoc.2138.
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