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May 2019

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Circadian Gene Profiling in Laser Capture Microdissected Mouse Club Cells
激光捕获显微解剖小鼠棒状细胞的昼夜节律基因分析   

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Abstract

Cell heterogeneity is high in tissues like lung. Research conducted on pure population of cells usually offers more insights than bulk tissues, such as circadian clock work. In this protocol, we provide a detailed work flow on how to do circadian clock study by RNA seq in laser capture micro-dissected mouse lung club cells. The method uses frozen tissues and is highly reproduciable.

Keywords: Club cell (棒状细胞), Lung (肺), Circadian clock (生物钟), RNA seq (RNA测序), Laser capture micro-dissection (激光捕获显微解剖)

Background

Circadian clock is found all over the body and in the lung it is shown to strongly oscillate in Club cells (Gibbs et al., 2009 and 2014). Whole lung circadian gene profiling is carried before in mice and rat (Sukumaran et al., 2011; Zhang et al., 2014). However, at least 15% of those circadian genes are coming from leukocytes (Haspel et al., 2014). It is useful but difficult to get cell type specific circadian gene profiling, given the high heterogeneity of cell population in tissues. Tissue digestion followed by flow cytometry sorting is usually to solve this problem but it is not clearly whether gene expression will change during this process. An alternative way is to use laser capture microdissection to take target cells from fixed tissues, Note that this could lead to comprised cell population purity. Specific cell population can be identified by antibody staining or anatomic information. In terms of Club cells in lung, they are known to be enriched in distal bronchiolar epithelium. A protocol showing how to do microarray using RNA from Club cells by laser capture (PixCell II Laser Capture Microdissection system) was published more than 10 years before and there are some advances in the technique (Betsuyaku and Senior, 2004; Cummings et al., 2011). Here I present a detailed protocol on circadian gene profiling in Club cell RNA by laser capture microdissection in Leica LMD6500 system. It covers from mouse tissue collection to get RNA samples ready for RNA seq library construction. Generally, to get good quality RNA, all tools and working areas should keep RNase contamination to the minimum (like using RNaseZAP solution to clean and keep sections dry in microdissection). For all the downstream steps, please refer to the published protocol by Jiajia Li, etc. (Li et al., 2015).

Materials and Reagents

  1. Smiths MedicalTM PortexTM Fine-Bore Polyethylene Tubing (Ref: 800/100/200, 0.58 mm ID and 0.96 mm OD)
  2. 23 G needles and 1 ml syringe
  3. PEN (polyethylene naphthalate) membrane slide (Leica, 11505158 ; Applied Biosystem, catalog number: LCM0522 ) and slide mail box
  4. Thin wall 0.5 ml Eppendorf tubes (Invitrogen, catalog number: Q32856 )
  5. Mice (C57BL or others)
  6. Phenobarbitone solution (for example, Pentoject, Animalcare limited, UK)
  7. 70% ethanol
  8. Dry ice or liquid nitrogen
  9. PBS tablet (Sigma, catalog number: P4417-100TAB )
  10. Sucrose
  11. RNaseZAP solution (Thermo Fisher, catalog number: AM9780 )
  12. Ethanol solutions (100%, 95%, 75%, 50%)
  13. Lysis buffer (supplied in kit) (Applied Biosystems, catalog number: KIT0204 )
  14. Picopure RNA isolation kit (Applied Biosystems, catalog number: KIT0204 )
  15. Qubit® RNA HS (High Sensitivity) assay kit (Invitrogen, catalog number: Q32852 )
  16. Agilent RNA 6000 Pico Kit (Agilent, catalog number: 5067-1511 )
  17. OCT (Optimal cutting temperature) compound solution (any brand, for example, Fisher Scientific, catalog number: 12678646 ) and PBS (see Recipes)

Equipment

  1. Qubit machine
  2. Light dark chamber
  3. Cryostat
  4. Forceps and scissors, fine string
  5. Leica Laser Microdissection Scope LMD 6500
  6. Tape station or Agilent 2100 Bioanalyzer
  7. -80 °C freezers

Procedure

  1. Lung tissue collection
    1. Entrained mice [7 day L/D (12 h light/12 h dark) followed by 1 day D/D (12 h dark/12 h dark)] are culled in D/D by either i.p. injection of phenobarbitone (200 μl/mouse) or neck dislocation. One mouse per time point is used and samples are collected every 4 h for 48 h. Once they are dead, take the bodies to the procedure area.
      Notes:
      1. In previous lung circadian gene profiling studies, the condition varies from L/D, D/D and L/L (12 h light/12 h light).
      2. The former method is preferred as sometimes tracheas can be damaged by neck dislocation.
      3. Though samples pooled from several mice (like n = 3) is used often in literature for this kind of study, the data from our study suggest that this may not be necessary. Sometimes, when mouse stock is limited, mice can be culled at different experiments (for example, only CT0-32 samples are collected in one experiment and the remaining is collected from another experiment). We don’t find big effect on the result; even tissues are collected almost 6 months apart.
    2. Spray mice with 70% ethanol and make a vertical cut in skin from abdomen to chin. Open the pleural cavity by piercing the pleural membrane and remove ribs above the lung. Lift and cut off thymus to expose trachea. The connective tissues surrounding trachea can then be peeled apart by blunt forceps.
    3. Use spring scissors to make a nick cut in the upper part of the trachea and insert the BAL lavage tube fitted with a 23 G needle to the trachea. Strings can be used to make a tie to hold the insert.
      Note: It is not absolute necessary to do this as forceps can be used instead. But it helps in later steps during lung removal.
    4. Fill about 0.8 ml OCT solutions [50% (v/v) OCT in PBS solution (made with DEPC H2O) with 5% sucrose] in a 1 ml syringe. And slowly inject the solution to inflate the lung through trachea.
    5. Remove the lung with the trachea closed with forceps or a string tie. Trim off the heart and thymus before freezing lungs in either dry ice or liquid nitrogen. For storage, the lungs covered with foil paper are put in -80 °C freezers.
      Note: Samples frozen in liquid nitrogen are difficult to cut as the texture becomes very crisp. RNA quality from dry ice and liquid nitrogen frozen samples is comparable, without clear change.

  2. Lung sectioning
    1. Take out the lungs and place them in the cryostat for 30 min before cutting.
    2. Mount the lung in vertical position and coronal sections are cut in 10 µm thickness, 3-4 sections per PEN membrane slide.
      Note: Either RNase free or normal PEN slide can be used. For the latter, PEN membrane can be cleaned by UV light exposure in cell culture hood for 30 min or with RNaseZap.
    3. It is all right to directly place the tissue sections into a slide from room temperature but once a section is on the slide, leave the slide in the cryostat. Examples of dissection areas are shown in the picture.
    4. Usually, 10 slides/mouse are cut for RNA sequence study (150-450 ng total RNA yield after microdissection.
      Note: The amount depends on chosen method to construct library for sequencing. For Illumina Trueseq kit, 100 ng total RNA is the minimum amount requirement. When an amplification step can be used, much less material is required, with total RNA even in picrogram range.
    5. Yield from 2-3 slides is enough for getting RNA in qPCR study.
    6. Place the slides in a closed slide mail box and store the slides in -80 °C freezers. It is recommended to finish the slides microdissection within 2 weeks.

  3. Laser capture microdissection
    1. Specimen slides were taken out of dry ice and quickly place into alcoholic solutions as below:
      100% ethanol for 1 min, 75% ethanol 3 dips, 50% ethanol 3 dips, 95% ethanol 30 s, 100% ethanol 30 s, 100% ethanol 2-5 min. For ethanol solutions, avoid leaving open too long to prevent water coming in from air and change new solutions after prolonged use. Chemicals can be used to further dry the ethanol. It is essential to keep last ethanol solution 100%, as RNase can not work without water.
      Notes:
      1. Cresyl violet solution in ethanol can be used to stain sections but not necessary here. The bronchiolar structure is easy to recognise under light microscope without any staining.
      2. The aim of this process is to dissolve OCT in water and make the sections dry in the end to prevent RNase activity.
    2. Leica LMD 6500 laser capture microdissection machine is used. After slides being dried down, they are placed in the slide holder. For tissue collection, 0.5 ml thin wall PCR tubes are used in the tissue collector, with 30 µl lysis buffer added (add DTT before use according to the kit protocol, Picopure RNA isolation kit, Life Technologies).
    3. Inspect the specimen to look for the bronchiolar airways under the microscope with 6.7x or 10x magnifications. Bronchiolar epithelium was cut off from junction between terminal bronchiole and alveolar ducts, proximally ~200 μm along the bronchiole. The laser setting should be tested beforehand. The microdissection view and laser setting used in our lab is shown in the picture below (Figure 1).


      Figure 1. Illustration of laser capture of distal bronchiolar epithelial cells. The view is under 6.7x magnifications with 2 bronchiolar airways shown in the middle in A. B. shows how the cutting line is drawn and C. shows what the tissue ie like after cutting. Laser setting is shown in D.

    4. Check the collection solution about every 1 h and add another 30 µl of lysis buffer or DEPC H2O before the solution on the cap running dry. It is recommended to use 2 or 3 collection tubes for 1 sample to avoid the solution becoming too thick after long microdissection process. These tubes for the same sample can be pooled when it comes to RNA purification.
      Note: For Picopure RNA extraction kit, it is recommended to incubate samples at 42 °C for 30 min first to fully lysis the tissue. This step can be done either after immediate dissection or just before RNA extraction.
    5. It usually takes 5-8 h to manually dissect 10 slides. In the end, put the tube onto dry ice and the samples can be stored for long term in -80 °C.
      Notes:
      1. There is an option of automatic cutting after selecting multiple target areas. However, it is not uncommon to find target sections ‘hanging’ on the slide instead of falling off after closed cutting. In this case, a manual point shooting method is used to cut off target areas.
      2. Each slide takes 30-45 min to microdissect. Both coarse and fine stage movement can be used during microdissection. It is recommended to inspect the sections again with eyes under microscope after dissection of each slide to avoid missing target areas.

  4. RNA extraction and study
    Picopure RNA extraction kit from Life Technologies to extract the RNA is used according to enclosed protocol. Among the extraction process, DNA on column digestion is performed as described in the protocol. Final RNA is eluted in 20 μl volume and low RNA binding Eppendorf tube is recommend to hold the samples.
    Note: RNeasy micro kit from Qigen and EZNA microelute total RNA kit from VWR can also be used. It is also possible to do the extraction glycogen precipitation without any kit but it could take much longer time to do.
      For quantification, it is recommended to take 1 μl of RNA and dilute 1:5 in H2O. From this, use 1 μl to quantify RNA amount in Qubit® RNA HS (High Sensitivity) assay. The typical concentration is above 10 ng/μl. The remaining can be saved for qPCR validation of final RNA seq data. For quality assessment of RNA intactness, use Agilent RNA 6000 Pico Kit or Tape station. The RIN number is usually around 6-8. If the value is too low, new mouse lung samples should be collected to start the process from beginning again.
    Notes:
    1. When the concentration is measured in a normal NanoDrop machine, the concentration can be very inaccurate. Successful qPCR application is done with laser capture microddisected materials from just 2 slides when the concentration is in lower than 0 ng/μl range by NanoDrop.
    2. If the amount is too low, more slides can be used to microdissect and use this RNA solution to elute or pool the RNA together in the end. Also check Step A3 about an amplification step. But it is recommended to avoid this situation by collecting as much materials as possible in first place.
    3. Lung is known to have high levels of RNase. In other tissues, like brain, RIN number can be as high as 8-9.
    Typical RNA quality, qPCR result showing enrichment of Club cells and RNA seq result showing circadian gene expression are shown in figure below (Figure 2).


    Figure 2. Typical RNA quality, qPCR and RNA seq result. A. Tape station showing RNA quality. B. and C. Enrichment of Club cell marker and little endothelial cell contamination shown by qPCR. D. and E. Bmal1 and Per3 gene expression profile from RNA seq results on laser capture microdisssected Club cells.

Data analysis

Study results using this method is published on FASEB journal (Zhang et al., 2019).

Recipes

  1. OCT (Optimal cutting temperature) compound solution and PBS
    1. Solutions (50% (v/v) OCT) in 1x PBS solution (made with PBS tablet in DEPC H2O) with 5% sucrose
    2. Dissolve sucrose first before adding OCT and this will make sucrose easier to dissolve

Acknowledgments

The authors thank BBSRC, UK for funding of this work and the method is based on Betsuyaku and Senior (2004) and Cummings et al. (2011).

Competing interests

The authors don’t have any competing interests.

Ethics

All experiments on animals were carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act of 1986 and European Directive 2010/63/EU, following approval by the University of Manchester Animal Welfare and Ethical Review Body.

References

  1. Betsuyaku, T. and Senior, R. M. (2004). Laser capture microdissection and mRNA characterization of mouse airway epithelium: methodological considerations. Micron 35(4): 229-234.
  2. Cummings, M., McGinley, C. V., Wilkinson, N., Field, S. L., Duffy, S. R. and Orsi, N. M. (2011). A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue. Anal Biochem 416(1): 123-125.
  3. Gibbs, J. E., Beesley, S., Plumb, J., Singh, D., Farrow, S., Ray, D. W. and Loudon, A. S. (2009). Circadian timing in the lung; a specific role for bronchiolar epithelial cells. Endocrinology 150(1): 268-276.
  4. Gibbs, J., Ince, L., Matthews, L., Mei, J., Bell, T., Yang, N., Saer, B., Begley, N., Poolman, T., Pariollaud, M., Farrow, S., DeMayo, F., Hussell, T., Worthen, G. S., Ray, D. and Loudon, A. (2014). An epithelial circadian clock controls pulmonary inflammation and glucocorticoid action. Nat Med 20(8): 919-926.
  5. Haspel, J. A., Chettimada, S., Shaik, R. S., Chu, J. H., Raby, B. A., Cernadas, M., Carey, V., Process, V., Hunninghake, G. M., Ifedigbo, E., Lederer, J. A., Englert, J., Pelton, A., Coronata, A., Fredenburgh, L. E. and Choi, A. M. (2014). Circadian rhythm reprogramming during lung inflammation. Nat Commun 5: 4753.
  6. Li, J., Grant, G. R., Hogenesch, J. B. and Hughes, M. E. (2015). Considerations for RNA-seq analysis of circadian rhythms. Methods Enzymol 551: 349-367.
  7. Sukumaran, S., Jusko, W. J., Dubois, D. C. and Almon, R. R. (2011). Light-dark oscillations in the lung transcriptome: implications for lung homeostasis, repair, metabolism, disease, and drug action. J Appl Physiol (1985) 110(6): 1732-1747.
  8. Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E. and Hogenesch, J. B. (2014). A circadian gene expression atlas in mammals: implications for biology and medicine. Proc Natl Acad Sci U S A 111(45): 16219-16224.

简介

[摘要] 在肺等组织中细胞异质性很高。对纯细胞群体的研究通常比诸如昼夜节律的大块组织提供更多的见解。在本协议中,我们提供了有关如何进行昼夜节律研究的详细工作流程。用RNA seq技术在激光捕获的小鼠肺部微小组织中进行捕获。该方法使用冷冻的组织,具有很高的重现性。

[背景 ] 昼夜节律钟遍布全身,并且在肺中显示出在Club细胞中强烈振荡(Gibbs 等人,2009年和2014年),之前在小鼠和大鼠中进行了整个肺节律基因分析(Sukumaran 等人)。。,2011 ; 张等人。2014)。然而,至少15 Pasento那些昼夜基因是即将从白细胞(Haspel的等人。2014)。它是有用的,但很难得到小区类型特定的节律基因分析,鉴于组织中细胞群体的异质性很高。通常通过组织消化和流式细胞仪分选来解决这个问题,但尚不清楚在此过程中基因表达是否会发生变化。另一种方法是使用激光捕获显微解剖从靶标中提取靶细胞固定的组织,请注意这可能会导致特定的细胞群纯度。特定的细胞群可以通过抗体染色或解剖学信息来识别。就肺中的Club细胞而言,已知它们富含 十年前发表了一项协议,展示了如何使用激光捕获(PixCell II激光捕获显微切割系统)的Club细胞RNA进行微阵列操作的协议,该技术已有一些进展(Betsuyaku and Senior,2004 ; Cummings 等人。,2011)。在这里,我提交一份详细的协议在节律基因谱在俱乐部细胞RNA激光捕获显微切割在徕卡LMD6500系统,它涵盖了从小鼠组织收集至获取RNA样品准备好RNA测序文库的构建。一般情况下,要为了获得高质量的RNA,所有工具和工作区域都应将RNase污染降至最低(例如使用RNaseZAP 溶液清洁显微切片并使切片保持干燥)。有关所有下游步骤,请参阅Li Jiajia Li 等发表的方案。(Li et al 。,2015)。

关键字:棒状细胞, 肺, 生物钟, RNA测序, 激光捕获显微解剖

材料和试剂


 


史密斯医疗TM Portex TM 细口径的聚乙烯管(参考:800/100/200,0.58 毫米内径0.96 毫米外径)
23 G针和1 ml注射器
PEN(聚萘二甲酸乙二醇酯)膜片(徕卡,11505158 ;应用乙Iosystem,目录号:LCM0522 )和幻灯片信箱
薄壁0.5 ml Eppendorf管(Invitrogen,目录号:Q32856 )。
小鼠(C57BL或其他)
苯巴比妥溶液(例如,Pentoject ,Animalcare limited,英国)
70%乙醇
干冰或液氮
PBS片剂(Sigma,目录号:P4417-100TAB)
蔗糖
RNaseZAP 解决方案(赛默飞世尔,目录号:AM9780 )
乙醇溶液(100%,95%,75%,50%)
裂解缓冲液(试剂盒中提供)(Applied Biosystems,目录号:KIT0204 )。
Picopure RNA分离试剂盒(Applied Biosystems,目录号:KIT0204 )。
量子® RNA HS(高灵敏度)测定试剂盒(Invitrogen公司,目录号:Q32852)
Agilent RNA 6000 Pico试剂盒(Agilent,目录号:5067-1511 )。
OCT(最佳切割温度)复合溶液(任何品牌,例如Fisher Scientific,目录号:12678646)和PBS(请参见食谱)
 


配套设备


 


Qubit机器
暗室
低温恒温器
镊子和剪刀,细绳
徕卡激光显微切割镜LMD 6500
磁带站或Agilent 2100生物分析仪
-80 °C 冰柜
 


程序


 


肺组织收集离子
夹带的小鼠[ 第7天L / d(12 小时光照/ 12 ,随后1天d / d(12小时黑暗))小时黑暗/ 12 小时黑暗)] 被屠宰在d / d通过要么叶。注射苯巴比妥(200 μl /只小鼠或颈部脱臼。每个时间点使用一只小鼠,每4 h 收集样品48 h,一旦它们死了,将尸体移至手术区域。
注意事项:


在先前的肺昼夜节律基因分析研究中,病情因L / D,D / D和L / L(12 h光照/ 12 h光照)而异。
首选前一种方法,因为有时气管可因颈部脱位而受损。
尽管文献中经常使用从几只小鼠(如n = 3)中收集的样本进行此类研究,但我们的研究数据表明这可能不是必需的。当小鼠存量有限时,可以以不同的方式淘汰小鼠实验(例如,在一个实验中仅收集CT0-32样本,而从另一个实验中收集其余样本)。我们对结果没有太大影响;甚至组织也相距近6个月。
用70%的乙醇喷洒小鼠,从腹部到下巴垂直切开皮肤,刺穿胸膜以打开胸膜腔并去除肺上方的肋骨,提起并切断胸腺以暴露气管,然后可以围绕气管结缔组织被钝钳剥开。
用弹簧剪刀在气管上部切开一个缺口,然后将装有23 G针的BAL灌洗管插入气管中。可用细绳系结以固定插入物。
注意:这样做不是绝对必要的,因为可以使用镊子代替,但这在肺切除的后续步骤中很有帮助。


填充约0.8μm 升OCT 溶液[ 50%(V / V)OCT在PBS溶液(用DEPCħ制成2 O)用5%的蔗糖] ,在1 毫升注射器。并缓慢注入溶液通过气管膨胀肺。
取出肺部,用镊子或扎带闭合气管。修剪心脏和胸腺,然后在干冰或液氮中冷冻肺部。用箔纸盖住的肺部放入-80 °C的冷冻器进行保存。
注意:液氮中冷冻的样品由于质地变得非常脆而难以切割,干冰和液氮冷冻样品中的RNA质量相当,没有明显变化。


 


肺切片
切除前,取出肺部并将其放在低温恒温器中30 分钟。
将肺垂直安装,将冠状切片切成10 µm的厚度,每个PEN膜玻片3-4个切片。
注意:可以使用无RNase的PEN玻片或普通的PEN玻片。对于后者,可以通过在细胞培养罩中暴露30分钟的紫外线或使用RNaseZap 清洁PEN膜。


可以从室温直接将组织切片直接放入载玻片中,但是一旦将切片放在载玻片上,就将其留在低温恒温器中。解剖区域的示例如图所示。
通常,每只小鼠要切10张玻片用于RNA序列研究(显微解剖后总RNA产量为150-450 ng)。
注意:用量取决于构建测序文库的方法。对于Illumina Trueseq 试剂盒,最低RNA量要求为100 ng。使用扩增步骤时,所需材料少得多,即使在象形图范围内也需要总RNA 。


2-3张载玻片的产量足以在qPCR研究中获得RNA。
将幻灯片放在封闭的幻灯片邮箱中,并将幻灯片存储在-80中 °C冰柜,建议在2周内完成玻片显微切割。
 


激光捕获显微切割
将标本载玻片从干冰中取出,并迅速放入酒精溶液中,如下所示:
100%乙醇1 分钟,75%乙醇3次浸入,50%乙醇3次浸入,95%乙醇30 秒,100%乙醇30 秒,100%乙醇2-5 分钟。对于乙醇溶液,请避免将其放置太长时间防止水从空气中进入并在长时间使用后更换新溶液。可使用化学物质进一步干燥乙醇。必须使最后的乙醇溶液保持100%,因为RNase 不能没有水就不能工作。


注意事项:


乙醇中的甲酚紫溶液可以用来染色切片,但在这里不是必需的。细支气管的结构很容易在光学显微镜下识别而没有任何染色。
此过程的目的是将OCT溶解在水中并使切片最终干燥以防止RNase活性。
徕卡LMD 6500激光捕获显微切割机是采用后滑轨干燥下来,他们被放置在片夹,对于组织收集,0.5 毫升薄壁PCR管用于在组织收集,用30 Myueru裂解液添加(广告d根据试剂盒协议,使用Picture RNA分离试剂盒,Life Technologies ,然后使用DTT 。
检查样本,寻找支气管航空公司在显微镜下W¯¯ Ith的6.7 X 或10倍放大倍率。支气管上皮被切断结终末细支气管和肺泡管道之间,近侧〜 200 Myuemu 沿细支气管。激光设置应该是测试版事前下图显示了我们实验室使用的显微剖视图和激光设置(图1)。
 






图1 ,图示激光捕获远端细支气管上皮细胞组成。该视图是在6.7 X 放大倍率随着2个细支气管航空公司显示在中间在A,B ,展示了如何切割线绘制与C ,显示了组织即等之后激光设置在D中显示。


 


检查收集解决方案约每1个小时,增加30 MYU 大号裂解缓冲区或DEPC ^ h 2 Ø之前的解决方案在帽上干运行,建议使用2个或3个收集管对于1个样品,以避免解决方案过于经过长时间的显微解剖后,这些样品管的厚度会增加。
注:对于Picopure RNA提取试剂盒,建议孵化样品在42 ℃,为30分钟,首个完全裂解组织这一步既可以做即时夹层或者只是RNA提取之前之后。  


通常需要5-8它h至手动解剖10幻灯片。最后,将管子放干冰上,样品可以储存长期在-80 ℃下。
注意事项:


选择多个目标区域后可以选择自动切割,但是在闭合的切割位置上发现目标部分``悬挂''而不是掉下来并不罕见,这种情况下,使用手动点射击方法进行切割偏离目标区域。
每个载玻片需要30-45 分钟的时间进行显微切割。在显微切割过程中,可以同时进行粗略和精细的移动。建议在解剖每个载玻片后用显微镜再次检查切片,以免丢失目标区域。
 


 


 


RNA提取和研究
Picopure RNA提取试剂盒自Life Technologies抽提RNA用于根据封闭协议。在提取过程中,DNA柱上消化执行。如描述的协议。最终RNA洗脱在20 Myueru 容量低RNA结合的Eppendorf管建议拿着样品。


注:微的RNeasy试剂盒气根和EZNA Microelute 总RNA试剂盒VWR也可以用它也可以做提取糖原降水没有任何工具,但它可能需要更长的时间待办事宜。


量化方法,建议以1 Myueru RNA的稀释1 :5在H 2 O.由此看来,使用1 Myueru 量化RNA量量子比特® 。RNA HS(高灵敏度)分析典型的浓度高于10 伍/ μl 。剩余的可以保存用于最终RNA seq数据的qPCR验证。对于RNA完整性的质量评估,请使用Agilent RNA 6000 Pico试剂盒或Tape station.RIN值通常在6-8左右。如果值太低,应收集新的小鼠肺部样本以重新开始该过程。 


注意小号:


当浓度测定在一个正常的纳米d 罗普机,其浓度可以是非常不准确的。成功的QPCR应用做随着激光捕获Microddisected 材料从仅有2幻灯片时的浓度是在低于0纳克/ Myueru 范围内的纳米d 罗普。
如果量太低,可以使用更多的玻片进行显微解剖,最后使用该RNA溶液将RNA洗脱或汇集在一起,并检查步骤A3的扩增步骤,但是建议通过收集以下步骤避免这种情况首先,尽可能多的材料。
已知肺中的RNase含量很高,在其他组织(如脑)中,RIN值可高达8-9。
下图显示了典型的RNA质量,显示Club细胞富集的qPCR结果和显示昼夜节律基因表达的RNA seq结果(图2)。


 


 


D:\重新格式化\ 2020-3-2 \ 1902957--1365张振光837218 \无花果jpg \ fig2.jpg


图2 。典型的RNA质量,QPCR和RNA SEQ结果。阿。磁带Station显示RNA质量。乙。与C 。富集所示QPCR。d俱乐部细胞标记和小血管内皮细胞的污染。和e 。BMAL1与Per3基因表达RNA seq的结果分析了激光捕获的显微解剖的Club细胞。


 


资料分析


 


使用这种方法的研究结果发表在FASEB期刊上(Zhang 等,2019)。


 


菜谱


 


OCT(最佳切割温度)复合溶液和PBS
含5%蔗糖的溶液(1x PBS溶液中的50%(v / v)OCT (由PBS片剂在DEPC H 2 O中制得))
在添加OCT之前先溶解蔗糖,这将使蔗糖更易于溶解
 


致谢


 


作者感谢BBSRC的,英国对于这种资助工作和方法是基于Betsuyaku和高级(2004年 )和卡明斯等人。(2011)。


 


竞争利益


 


作者没有任何竞争利益。


 


 


 


道德规范


 


经曼彻斯特大学动物福利和伦理审查机构批准,所有动物实验均根据1986年英国内政部动物(科学程序)法和欧洲指令2010/63 / EU进行。


 


参考小号


 


Betsuyaku ,T.和Senior,RM(2004)。小鼠气道上皮的激光捕获显微切割和mRNA表征:方法上的考虑。微米35(4):229-234。
Cummings,M.,McGinley,CV,Wilkinson,N.,Field,SL,Duffy,SR和Orsi ,NM(2011)。用于子宫内膜癌组织激光捕获显微切割的稳健的RNA完整性染色方案 .Anal Biochem 416( 1):123-125。
吉布斯,JE,比斯利,S.,铅锤,J.,辛格,D.,法罗,S.,雷,DW及劳登,AS(2009)。昼夜定时在肺;.特定角色对于细支气管上皮细胞内分泌学150(1):268-276。
Gibbs,J.,Ince,L.,Matthews,L.,Mei,J.,Bell,T.,Yang,N.,Saer ,B.,Begley,N.,Poolman ,T.,Pariollaud ,M., Farrow,S.,DeMayo ,F.,Hussell ,T.,Worthen,GS,Ray,D.和Loudon,A.(2014)。上皮生物钟控制肺部炎症和糖皮质激素的作用 .Nat Med 20(8): 919-926。
Haspel,JA,Chettimada ,S. ,Shaik ,RS,Chu,JH,Raby,BA,Cernadas ,M.,Carey,V.,Process,V.,Hunninghake ,GM,Ifedigbo ,E.,Lederer,JA,Englert ,J.,佩尔顿,A.,Coronata ,A.,Fredenburgh ,LE和彩,AM(2014)。昼夜节律重新编程期间肺部炎症。纳特COMMUN 5:4753。
Li,J.,Grant,GR,Hogenesch ,JB和Hughes,ME(2015)。昼夜节律RNA-seq分析的注意事项。方法Enzymol 551:349-367。
Sukumaran,S.,Jusko ,WJ,Dubois,DC和Almon,RR(2011)。肺转录组中的明暗振荡:对肺稳态,修复,代谢,疾病和药物作用的影响。J Appl Physiol (1985)。110(6):1732-1747。
Zhang,R.,Lahens ,NF,Ballance,HI,Hughes,ME和Hogenesch ,JB(2014)。哺乳动物中的昼夜节律基因表达图谱:对生物学和医学的影响 .Proc Natl Acad Sci USA 111(45):16219- 16224。
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引用:Zhang, Z. and Loudon, A. (2020). Circadian Gene Profiling in Laser Capture Microdissected Mouse Club Cells. Bio-protocol 10(8): e3590. DOI: 10.21769/BioProtoc.3590.
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