本文描述了一种详细和高效的RNA 原位杂交方案,特别是对于低水平表达的气味受体(OR)基因以及其他基因,在使用地高辛(DIG)标记或生物素标记的探针的昆虫天线中。
昆虫已经发展了复杂的嗅觉接收系统来感测外源化学信号。这些化学信号由嗅觉受体神经元(ORNs)转导,其位于天线的头发状结构(称为化学感光体)中。在ORN的膜上,认为气味受体(ORs)参与气味编码。因此,能够识别定位于ORN的基因是识别OR基因所必需的,为进一步的功能性原位研究提供了基础。昆虫天线中特异性OR的RNA表达水平非常低,用于组织学的保存昆虫组织具有挑战性。因此,使用RNA 原位杂交难以将OR定位到特定类型的感觉器。在本文中,介绍了详细和高效的RNA 原位杂交方案,特别是对昆虫的低表达OR基因。另外还有一个具体的OR 基因哇通过使用共表达受体基因Orco进行双色荧光原位杂交实验鉴定 。
作为最重要的化学感觉器官的昆虫天线被许多由嗅觉受体神经元(ORN)支配的头发状结构(称为感觉)覆盖。昆虫ORNs的膜,气味受体(ORS),一种类型的包含七个跨膜结构域的蛋白质,与共同受体(ORCO)以形成杂聚体表示其功能如同一个加臭剂门控离子通道1,2,3。不同的OR响应化学化合物4,5,6的不同组合。
蝗虫( Locusta migratoria )主要依靠嗅觉信号触发重要行为7 。蝗虫OR是理解分子嗅觉机制的关键因素。将特定的蝗虫OR基因定位到a的神经元通过RNA的形态特异性感觉类型原位杂交(RNA ISH)是探索ORs功能的第一步。
RNA ISH使用标记的互补RNA探针来测量和定位组织,细胞或整个原位部分的特异性RNA序列,从而提供对生理过程和疾病发病机理的见解。地高辛标记(DIG标记)和生物素标记的RNA探针已被广泛用于RNA杂交。用地高辛-11-UTP或生物素-16-UTP进行RNA标记可以通过SP6和T7 RNA聚合酶的体外转录来制备。 DIG-和生物素标记的RNA探针具有以下优点:非放射性;安全;稳定;高度敏感;高度特异性并且使用PCR和体外转录容易产生。 DIG-和生物素标记的RNA探针可以发色和荧光检测。 DIG标记的RNA探针可以用抗地高辛碱碱检测使用光学显微镜或2位可以用高度敏感的化学发光底物硝基蓝四唑氯化物/ 5-溴-4-氯-3-吲哚基 – 磷酸酯甲苯胺盐(NBT / BCIP)可视化的ne磷酸酶(AP)共轭抗体使用共聚焦显微镜与4-氯-2-甲基苯并噻唑半氯化锌盐(Fast Red)偶合的3-羟基-3-萘甲酸-2'-苯基苯胺磷酸盐(HNPP)。可以用抗生物素链霉亲和素马萝卜过氧化物酶(HRP) – 共轭抗体检测生物素标记的RNA探针,该抗体可以使用共聚焦显微镜用荧光素 – 酪酰胺显色。因此,可以进行双色荧光原位杂交以使用DIG-和生物素标记的RNA探针在一个切片中检测两个靶基因。
具有DIG和/或生物素标记的探针的RNA ISH已被成功地用于定位嗅相关基因,如OR ,离子型受体,加味剂结合蛋白和感觉神经元的膜蛋白,在昆虫天线,但不限于, 黑腹果蝇 , 冈比亚按蚊 ,L.飞蝗和沙漠蝗虫Schistocera蝗 8,9,10,11,12,13,14,15,16。然而,为昆虫ORs进行RNA ISH时有两个实质性的挑战:(1)OR基因( ORco除外)以低水平表达,仅在少数细胞中表达,使信号检测非常困难,(2)保存昆虫组织组织学,使得形态被保留并且背景噪声低,可能是具有挑战性的。在本文中,详细和有效的方案描述了用于在昆虫中定位OR基因的RNA ISH提出了天线,包括显色和Tyramide信号放大(TSA)检测。
这是很难执行RNA ISH本地化或昆虫触角基因,因为的表达水平或基因除了ORCO,是非常低的和保存的组织学切片的昆虫触角是非常困难的。另外TSA检测也很棘手。为解决这些问题,应采取以下措施。天线选自具有薄且软的触角表皮的新蜕皮成虫蝗虫,其保持其在幻灯片上的形态。将冷冻样品切成12μm厚的切片。使用高灵敏度和特异性的生物素和DIG标记的探针。洗涤剂如Tween-20和Triton X-100用于在?…
The authors have nothing to disclose.
这项工作得到国家自然科学基金资助项目(No.1472037)的支持。本文中提及的商品名称或商业产品仅用于提供具体信息,并不意味着推荐。
Materials | |||
2×TSINGKETM Master Mix | TSINGKE, China | TSE004 | |
RNase-free H2O | TIANGEN, China | RT121-02 | |
REGULAR AGAROSE G-10 | BIOWEST, SPAIN | 91622 | |
Binding buffer | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
Balance buffer | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
Wash solution | TIANgel Midi Purification Kit, TIANGEN, China | DP209-02 | |
T Vector | Promega, USA | A362A | |
T4 DNA Ligase | Promega, USA | M180A | |
Escherichia coli DH5α | TIANGEN, China | CB101 | |
Ampicillin | Sigma, USA | A-6140 | |
Isopropyl β-D-1-thiogalactopyranoside | Inalco, USA | 1758-1400 | |
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside | SBS Genetech, China | GX1-500 | |
Nco I | BioLabs, New England | R0193S | |
Spe I | BioLabs, New England | R0133M | |
DIG RNA Labeling Kit | Roche, Switzerland | 11175025910 | |
Biotin RNA Labeling Kit | Roche, Switzerland | 11685597910 | |
DNase | DIG RNA Labeling Kit, Roche, Switzerland | 11175025910 | |
LiCl | Sinopharm, China | 10012718 | |
Ethanol | Sinopharm, China | 10009257 | |
Acetic acid | BEIJING CHEMICAL REGENTS COMPANY, China | 10000292 | |
Tissue-Tek O.C.T. Compound | Sakura Finetek Europe, Zoeterwoude, Netherlands | 4583 | |
Slides | TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China | FISH0010 | |
HCl | Sinopharm, China | 80070591 | |
Millex | Millipore, USA | SLGP033RS | |
Tween 20 | AMRESCO, USA | 0777-500ML | |
Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt | Roche, Switzerland | 11175041910 | |
Glycerol | Sinopharm, China | 10010618 | |
Name | Company | Catalog Number | Comments |
Solutions | |||
1×Tris-acetate-EDTA | Sigma, USA | V900483-1KG | 0.04mol/L Tris-Base |
1×Tris-acetate-EDTA | BEIJING CHEMICAL REGENTS COMPANY, China | 10000292 | 0.12%acetic acid |
1×Tris-acetate-EDTA | Sigma, USA | 03677 | Ethylenediaminetetraacetic acid disodium salt (EDTA) |
Luria-Bertani (LB) liquid medium | Sinopharm, China | 10019392 | 10g/L NaCl |
Luria-Bertani (LB) liquid medium | MERCK, Germany | VM335231 | 10g/L Peptone from casein (Tryptone) |
Luria-Bertani (LB) liquid medium | MERCK, Germany | VM361526 | 5g/L Yeast extract |
LB solid substrate plate | Sinopharm, China | 10019392 | 10g/L NaCl |
LB solid substrate plate | MERCK, Germany | VM335231 | 10g/L Peptone from casein (Tryptone) |
LB solid substrate plate | MERCK, Germany | VM361526 | 5g/L Yeast extract |
LB solid substrate plate | WISENT ING, Canada | 800-010-CG | 15g/L Agar Bacteriological Grade |
10×phosphate buffer saline (pH7.1) | Sinopharm, China | 10019392 | 8.5%NaCl |
10×phosphate buffer saline (pH7.1) | Sigma, USA | V900041-500G | 14mM KH2PO4 |
10×phosphate buffer saline (pH7.1) | Sigma, USA | V900268-500G | 80mM Na2HPO4 |
10×Tris buffered saline (pH7.5) | Sigma, USA | V900483-1KG | 1M Tris-Base |
10×Tris buffered saline (pH7.5) | Sinopharm, China | 10019392 | 1.5M NaCl |
Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sigma, USA | V900483-1KG | 100mM Tris-Base |
Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sinopharm, China | 10019392 | 100mM NaCl |
Detection Buffer (DAP) chromogenic detection pH9.5 TSA detection pH8.0 | Sigma, USA | V900020-500G | 50mM MgCl2·6H2O |
20×saline-sodium citrate (pH7.0) | Sinopharm, China | 10019392 | 3M NaCl |
20×saline-sodium citrate (pH7.0) | Sigma, USA | V900095-500G | 0.3M Na-Citrate |
4% paraformaldehyde solution (pH9.5) | Sigma, USA | V900894-100G | 4% paraformaldehyde |
4% paraformaldehyde solution (pH9.5) | Sigma, USA | V900182-500G | 0.1M NaHCO3 |
Sodium Carbonate Buffer (pH10.2) | Sigma, USA | V900182-500G | 80mM NaHCO3 |
Sodium Carbonate Buffer (pH10.2) | Sigma, USA | S7795-500G | 120mM Na2CO3 |
Formamide Solution (pH10.2) | MPBIO, USA | FORMD002 | 50% Deionized Formamide |
Formamide Solution (pH10.2) | 5×saline-sodium citrate | ||
Blocking Buffer in Tris buffered saline | Roche, Switzerland | 11175041910 | 1% Blot |
Blocking Buffer in Tris buffered saline | AMRESCO, USA | 0694-500ML | 0.03% Triton X-100 |
Blocking Buffer in Tris buffered saline | 1×Tris buffered saline | ||
Alkaline phosphatase solution | Roche, Switzerland | 11175041910 | 1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody |
Alkaline phosphatase solution | Blocking Buffer in Tris buffered saline | ||
Alkaline phosphatase/ horse radish peroxidase solution | Roche, Switzerland | 11175041910 | 1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody |
Alkaline phosphatase/ horse radish peroxidase solution | TSA kit, Perkin Elmer, USA | NEL701A001KT | 1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody |
Alkaline phosphatase/ horse radish peroxidase solution | Blocking Buffer in Tris buffered saline | ||
Hybridization Buffer | MPBIO, USA | FORMD002 | 50% Deionized Formamide |
Hybridization Buffer | 2×saline-sodium citrate | ||
Hybridization Buffer | Sigma, USA | D8906-50G | 10% dextran sulphate |
Hybridization Buffer | invitrogen, USA | AM7119 | 20 µg/ml yeast t-RNA |
Hybridization Buffer | Sigma, USA | D3159-10G | 0.2 mg/ml herring sperm DNA |
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Roche, Switzerland | 11758888001 | 1% 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (10mg/ml) |
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Roche, Switzerland | 11758888001 | 1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25mg/ml) |
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate | Detection Buffer | ||
Tyramide signal amplification substrate | TSA kit, Perkin Elmer, USA | NEL701A001KT | 2% fluorescein-tyramides |
Tyramide signal amplification substrate | TSA kit, Perkin Elmer, USA | NEL701A001KT | Amplification Diluent |
Name | Company | Catalog Number | Comments |
Instrument | |||
Freezing microtome | Leica, Nussloch, Germany | Jung CM300 cryostat | |
Spectrophotometer | Thermo SCIENTIFIC, USA | NANODROP 2000 | |
Optical microscope | Olympus, Tokyo, Japan | Olympus IX71microscope | |
Confocal microscope | Olympus, Tokyo, Japan | Olympus BX45 confocal microscope |