This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.
Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs' membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.
Insect antennae, which are the most important chemosensory organs, are covered with many hair-like structures – called sensilla – that are innervated by Olfactory Receptor Neurons (ORNs). On the membrane of insect ORNs, Odorant Receptors (ORs), a type of protein containing seven transmembrane domains, are expressed with a coreceptor (ORco) to form a heteromer that functions as an odorant-gated ion channel1,2,3. Different ORs respond to different combinations of chemical compounds4,5,6.
Locusts (Locusta migratoria) mainly rely on olfactory cues to trigger important behaviors7. Locust ORs are key factors for understanding molecular olfactory mechanisms. Localizing a specific locust OR gene to the neuron of a morphologically specific sensillum type by RNA In Situ Hybridization (RNA ISH) is the first step in exploring the ORs function.
RNA ISH uses a labeled complementary RNA probe to measure and localize a specific RNA sequence in section of tissue, cells or whole mounts in situ, providing insights into physiological processes and disease pathogenesis. Digoxigenin-labeled (DIG-labeled) and biotin-labeled RNA probes have been widely used in RNA hybridization. RNA labeling with digoxigenin-11-UTP or biotin-16-UTP can be prepared by in vitro transcription with SP6 and T7 RNA polymerases. DIG- and biotin-labeled RNA probes have the following advantages: non-radioactive; safe; stable; highly sensitive; highly specific; and easy to produce using PCR and in vitro transcription. DIG- and biotin-labeled RNA probes can be chromogenically and fluorescently detected. DIG-labeled RNA probes can be detected with anti-digoxigenin Alkaline Phosphatase (AP)-conjugated antibodies that can be visualized either with the highly sensitive chemiluminescent substrates nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate toluidine salt (NBT/BCIP) using an optical microscope or with 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (HNPP) coupled with 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (Fast Red) using a confocal microscope. Biotin-labeled RNA probes can be detected with anti-biotin streptavidin Horse Radish Peroxidase (HRP)-conjugated antibodies that can be visualized with fluorescein-tyramides using a confocal microscope. Thus, double-color fluorescent in situ hybridization can be performed to detect two target genes in one slice using DIG- and biotin-labeled RNA probes.
RNA ISH with DIG- and/or biotin-labeled probes has been successfully used to localize olfactory-related genes, such as OR, ionotropic receptor, odorant-binding protein and sensory neuron membrane protein, in insect antennae of, but not limited to, Drosophila melanogaster, Anopheles gambiae, L. migratoria and the desert locust Schistocera gregaria8,9,10,11,12,13,14,15,16. However, there are two substantial challenges when performing RNA ISH for insect ORs: (1) OR genes (except ORco) are expressed at low levels and only in a few cells, making signal detection very difficult, and (2) preserving insect tissue for histology, such that the morphology is preserved and the background noise is low, can be challenging. In this paper a detailed and effective protocol describing RNA ISH for localizing OR genes in insect antennae is presented, including both chromogenic and Tyramide Signal Amplification (TSA) detection.
NOTE: To limit RNA degradation, prepare solutions using wet-autoclaved distilled water (at 121 °C for 60 min) and also wet-autoclave materials.
1. Preparation of RNA ISH Antisense and Sense Probes
2. Preparation of Cryostat Sections
3. Fixing Sections
4. Hybridization
5. Staining
6. Observation
With chromogenic detection, a small subset of the antennal cells in every adult antennal section was denoted by the DIG-labeled LmigOR1 and LmigOR2 antisense probes (Figure 3). RNA ISH on consecutive sections to localize LmigOR1 and LmigOR2 showed that antennal cells expressing the two genes were located in ORN clusters expressing LmigORco, indicating that the putative LmigOR1 and LmigOR2 were actually expressed in ORNs (Figure 4a-4d). Occasionally, labeled dendritic-like structures were visualized (Figure 4e-4f). LmigOR1 and LmigOR2 were both localized to neurons in the basiconic sensilla (Figure 5a-5b), but they were not co-expressed in individual sensilla, indicating that they were present in different basiconic sensillium subtypes (Figure 5c–5d).
In TSA detection, double-color fluorescent in situ hybridization showed that LmigOR1-expressing cells (red color) were located to ORN clusters expressing LmigORco (green color), indicating that the putative LmigOR1 was expressed in ORNs (Figure 6a). A close view of the boxed areas in Figure 6a are shown in Figure 6b. LmigOR1– expressing neurons (green color, Figure 7a) and LmigOR2-expressing neuron (red color, Figure 7b) were located to different basiconic sensillium subtypes (Figure 7c).
Figure 1: Preparation of Insect Antennal Sections for RNA In Situ Hybridization. (a) The freezing microtome; (b-c) The samples are embedded in freezing O.C.T. Compound. Please click here to view a larger version of this figure.
Figure 2: Experimental Items for RNA In Situ Hybridization. (a) The slide holder and the plastic container. (b) The humid box. (c) The rocker. (d) The membrane filter. (e) Confocal microscope. Please click here to view a larger version of this figure.
Figure 3: Cellular Localization of LmigOR1 & LmigOR2 in Olfactory Organs. (a) Overview of LmigOR1-expressing cells in a locust antennal segment. (b) Overview of LmigOR2-expressing cells in a locust antennal segment. Arrowheads indicate cells expressing LmigOR1 (a) and LmigOR2 (b). Scale bars = 100 µm (a and b). Figures were adapted from Xu et al.12 Please click here to view a larger version of this figure.
Figure 4: Neuronal Identity of Antennal Cells Expressing LmigORs by Chromogenic Detections. (a-b) The labeling pattern of LmigOR1 (a) and LmigORco (b) antisense probe on consecutive sections of locust antenna. (c-d) The labeling pattern of LmigORco (c) and LmigOR2 (d) antisense probe on consecutive sections of locust antenna. (e-f) Illustration of occasionally labeled dendritic-like structures (indicated by red arrows). Scale bars = 50 µm (a-d); 20 µm (e and f). Figures were adapted from Xu et al.12 Please click here to view a larger version of this figure.
Figure 5: LmigOR1 & LmigOR2 are Expressed in ORNs Housed in Basiconic Sensilla. (a-b) Basiconic sensillum housed ORNs expressing LmigOR1 (a) and LmigOR2 (b). (c-d) The expression of LmigOR1 and LmigOR2 in distinct subset of antennal ORNs was verified on consecutive sections (c-d). Arrowheads denote antennal cells expressing LmigOR1 (a, c) and LmigOR2 (b, d). Ba: basiconic sensillum. Scale bars = 20 µm (a and b); 50 µm (c and d). Figures were adapted from Xu et al.12 Please click here to view a larger version of this figure.
Figure 6: Neuronal Identity of Antennal Cell Expressing LmigOR1 by TSA Detections. (a) Two-color in situ hybridization was performed on longitudinal antennal section to illustrate the expression of LmigOR1 (Red) and LmigORco (Green). Localization of LmigOR1-expressing cells in cell clusters expressing LmigORco confirmed its neural identity. (b) Close view of boxed areas in a. Occasionally labeled dendritic like structures were indicated by arrow. Circled areas indicate ORNs cluster expressing LmigORco and sharing the same sensillum. Scale bars = 50 µm (a); 20 µm (b). Figures were adapted from Xu et al.12 Please click here to view a larger version of this figure.
Figure 7: LmigOR1 & LmigOR2 were Located to Different Basiconic Sensilla ORNs by TSA Detection. Fluorescent signals were visualized using detection systems, indicating LmigOR1-labeled neurons by green fluorescence (a) and LmigOR2 positive cells by red fluorescence (b) were expressed in different basiconic sensilla subtypes (c). Arrowheads denote antennal cells expressing LmigOR1 and LmigOR2. Scale bars = 20 µm. Figures were adapted from Xu et al.12 Please click here to view a larger version of this figure.
Name | Sequences |
Lmig OR1-probe-s | 5’-AAGGGGTGGGAGACGGCCTG-3’ |
Lmig OR1-probe-as | 5’-CAGCTCCTCCCCAACGACAGC-3’ |
Lmig OR2-probe-s | 5’-ATGGGTGAGCGTGGAGAGGC-3’ |
Lmig OR2-probe-as | 5’-GGTCATCGCTGTGGACGTGG-3’ |
Lmig Orco-probe-s | 5’-CTCGTCTGACAGCGTAACTCAC-3’ |
Lmig Orco-probe-as | 5’-AAGACGCAGAAGAGGAAGACCT-3’ |
Table 1: The Sequences of the PCR Primers.
It is hard to perform RNA ISH to localize OR genes in insect antennae because the expression levels of OR genes, except ORco, are very low and preserving histological slices of insect antennae is very difficult. In addition, TSA detection is also very tricky. To address these problems, the following measures should be taken. The antennae are selected from newly molting adult locusts that have thin and soft antennal cuticles, which maintain their morphology on the slide. The frozen samples are sectioned into 12 µm-thick slices. Highly sensitive and specific biotin- and DIG-labeled probes are used. Detergents, such as Tween-20 and Triton X-100, are used to decrease the background in many steps. In TSA detection, a highly expressed gene, relative to another lowly expressed gene, should be labeled with biotin-16-UTP. The slides should be observed as soon as possible because the fluorescent signals will quench quickly.
DIG- and biotin-labeled probes have the advantages of longer shelf lives, higher signal to noise ratios and better cellular resolutions than radioactive probes18,19. The protocol presented in this paper has some advantages over whole mount in situ hybridization. This protocol easily identifies the localizations of genes at the cellular level but cannot be easily used to investigate gene distribution patterns, which is more readily performed with whole mount in situ hybridization.
To identify an OR gene, two approaches were taken. One was the chromogenic detection in consecutive sections, and the other was fluorescent detection in one section. ORco is co-expressed with a specific OR as an ORN marker10,20,21. Cells expressing LmigOR1 or LmigOR2 were both located to clusters of LmigORco-expressing cells that unambiguously verified them as ORs (Figure 4 and Figure 6). Using the same approaches, we found that LmigOR1 and LmigOR2 are not co-expressed in one sensillum. The results of these two approaches are corroborative.
This protocol was also successfully used to localize LmigORco, SgreORco, SgreIR8a and SgreIR25a in locust antennae10,14. Recently LmigOR3 was localized to trichoid sensilla neurons in L. migratoria using the same protocol15. This protocol was used to localize two sensory neural membrane proteins SgreSNMP1 and SgreSNMP2 in the antennae of S. gregaria16. Thus, this protocol reliably localized chemosensory-related genes in locust antennae, which not only verified these candidate genes, but also localized these genes to specific cells housed in different types of sensilla.
In conclusion, this highly effective protocol of RNA ISH is specifically described to localize OR genes, as well as other genes expressed at low levels, in insect antennae.
The authors have nothing to disclose.
This work is supported by a grant from National Natural Science Foundation of China (No.31472037). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation.
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 |