드로소필라 늦은 푸파 간접 비행 근육 (IFM) 해부 : 높은 처리량 조직 수집을위한 방법

Abstract

출처: 카오, S. Y., 외. 오믹스 접근에 대한 드로소필라 멜라노가스터 비행 근육의 해부. J. 비스 익스펙. (2019).

Drosophila 비행 근육 근육 개발 및 생리학에 대 한 모델 시스템으로 사용 됩니다. 이 비디오는 성인의 비행 근육을 설명하고 pupae에서 RNA 염기서열에 적합한 간접 비행 근육을 개발하는 해부 프로토콜을 강조합니다.

Protocol

이 프로토콜은 카오 외에서발췌., Omics 접근에 대한 드로소필라 멜라 노가스터 비행 근육의 해부, J. Vis. Exp. (2019). 1. IFM 해부 후 48 h APF 두 #5 생물학 학년 집게, 미세 가위, 표준 유리 현미경 슬라이드, 이중 스틱 테이프, 파이펫, 파이펫 팁, 드라이 아이스 및 (RNA 응용 프로그램의 경우) 격리 시약 (재료의 표참조)을 포함하여 필요한 장비를…

Representative Results

그림 1: 48h APF 후 IFM의 해부. (A)이중 스틱 테이프에 강아지의 정렬. (B)푸팔 케이스로부터 푸파를 제거하고, 케이스를 등등(B’)으로 자르고,강아지(B’)를들어올리는 것으로. 도 2와동일하게 표시된 원 기호입니다. (C)버퍼에 강아지의 전송. (D)</…

Materials

60 mm culture dishes	Sigma-Aldrich	Z643084-600EA	Greiner dishes, 60 mm x 15 mM, vented
Cell phone camera, Samsung Galaxy S9	Samsung	SM-G960F/DS	used for photos not taken under a microscope
Double stick tape	Scotch/3M	3M ID 70005108587	Double-sided tape, available at most office supply handlers
Dumont #5 Forceps	Fine Science Tools	11252-20	Inox straight tip 11 cm forceps, Biology grade with 0.05 mm x 0.02 mm tip
fluorescent dissecting microscope camera, Leica DFC310 FX camera	Leica		www.leica-microsystems.com
Fluorescent dissecting microscope software, Leica Application Suite (LAS) version 4.0.0	Leica		www.leica-microsystems.com
Fluorescent dissecting microscope, Leica M165 FC	Leica		www.leica-microsystems.com
Fly: Bru1[M2]			Fly stock; This paper
Fly: Bru1[M3]			Fly stock; This paper
Fly: Mef2-GAL4	Bloomington Stock Center	BDSC:27390	Fly stock
Fly: salm[1]	Bloomington Stock Center	3274	Fly stock
Fly: salm[FRT]			Fly stock; see Spletter et al., Elife, 2018
Fly: UAS-Bru1IR	Vienna Drosophila Research Center	GD41568	Fly stock, RNAi hairpin
Fly: UAS-GFP::Gma	Bloomington Stock Center	BDSC:31776	Fly stock
Fly: UAS-mCD8a::GFP	Bloomington Stock Center	BDSC:5130	Fly stock
Fly: w[1118]	Bloomington Stock Center	3605	Fly stock
Fly: weeP26			Fly stock; see Clyne et al., Genetics, 2003
GFP detection reagent, GFP-Booster	ChromoTek	gba488-100
Glycogen	Invitrogen	10814-010
Image processing software, Photoshop CS6	Adobe		www.adobe.com
Isopropanol	Sigma-Aldrich	I9516-25ML	2-propanol
Method 1 (RNA isolation): TRIzol	Life Technologies	15596018	Guanidinium isothiocyanate and phenol monophasic solution
Method 2 (RNA isolation): Method 1 + TURBO DNA-free Kit	Invitrogen	AM1907	TRIzol isolation followed by treatment with a kit to remove DNA
Method 3 (RNA isolation): Direct-zol RNA Miniprep Plus Kit	Zymo Research	R2070S	RNA isolation in TRIzol, but over commercial columns instead of using phase separation. Recommended DNase treatment performed with Monarch Dnase I in Monarch DNase I Reaction buffer.
Method 4 (RNA isolation): RNeasy Plus Mini Kit	Qiagen	74134	We used the provided DNase treatment. IFM pellets were homogenized in RTL buffer as suggested for animal tissues.
Method 5 (RNA isolation): ReliaPrep RNA Tissue Miniprep System	Promega	Z6110	We applied the protocol for 'Purification of RNA from Fibrous Tissues'.
Method 6 (RNA isolation): Monarch Total RNA Miniprep Kit	New England Biolabs	T2010G	We applied the protocol for tissues/leukocytes and lysed in 300 µL of RNA Protection Reagent.
Microcentrifuge tubes	Thermo Fisher	AM12400	RNase-free Microfuge Tubes, 1.5 mL
Microscope slides	Thermo Fisher	12342108	Standard slides, uncharged, 1 mm
Paintbrush	Marabu	1910000000	Marabu Fino Round No. 0, or similar brush from any art supply
Paraformaldehyde	Sigma-Aldrich	158127
PBS buffer (1x)	Sigma-Aldrich	P4417	Phosphate buffered saline tablets for 1 L solutions, pH 7.4
PFA PureTip Pipette Tips	Elemental Scientific	ES-7000-0101	Optional substitute for standard pipette tips to reduce sample loss; 100 mL, 0.8 mm orifice
Pipette tips	Sigma-Aldrich	P5161	Universal 200 mL pipette tips
RNA concentration Approach 1 & RNA integrity traces, Bioanalyzer	Agilent Technologies	G2939BA
RNA concentration Approach 2, Nanodrop	Thermo Fisher	ND-2000
RNA concentration Approach 3, Qubit 4 Fluorometer	Invitrogen	Q33238
RNase A	Promega	A7937
RNase-free water, Diethyl pyrocarbonate (DEPC)	Sigma-Aldrich	D5758	DEPC treat water overnight and then autoclave, to remove all RNase.
RT Kit #1: Super Script III Reverse Transcriptase Kit	Invitrogen	18080-044	reverse transcription kit
RT Kit #2: LunaScript	New England Biolabs	E3010S	reverse transcription kit
RT Kit #3: QuantiNova Reverse Transcription Kit	Qiagen	205410	reverse transcription kit
Statistical software: GraphPad Prism	GraphPad Prism		www.graphpad.com
Statistical software: Microscoft Excel	Microsoft		Purchased as part of the bundle: Office Home & Student 2019
Table-top centrifuge	Eppendorf	5405000760	Eppendorf Centrifuge 5425 or equivalent
Tissue/ Kimwipes	Sigma-Aldrich	Z188956	Standard tissue wipes
Transfer pipette	Sigma-Aldrich	Z350796	Plastic pipette
Vannas spring scissors	Fine Science Tools	15000-00	3 mm cutting edge, tip diameter 0.05 mm, length 8 cm
Whatman paper	Sigma-Aldrich	1004-070	Filter paper circles, Grade 4, 70 mm