Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissectie: een methode voor high-throughput weefselverzameling

Abstract

Bron: Kao, S. Y., et al. Dissectie van Drosophila melanogaster Flight Muscles for Omics Approaches. J.Vis. Exp. (2019).

De Drosophila vluchtspieren worden gebruikt als een modelsysteem voor spierontwikkeling en fysiologie. Deze video beschrijft het vluchtspierstelsel van volwassenen en belicht een protocol om het ontwikkelen van indirecte vluchtspieren te ontleden die geschikt zijn voor RNA-sequencing van poppen.

Protocol

Dit protocol is een uittreksel uit Kao et al., Dissectie van Drosophila melanogaster Flight Muscles for Omics Approaches, J. Vis. Exp. (2019). 1. IFM Dissectie Na 48 h APF Monteer de benodigde apparatuur, waaronder twee #5 van biologische kwaliteit, fijne scharen, standaard glasmicroscoopglaasjes, dubbelstokband, pipet, pipettips, droogijs en (voor RNA-toepassingen) isolatiereagens (zie tabel met materialen). Koel de 1x PBS en microcentrifu…

Representative Results

Figuur 1: Dissectie van IFM’s na 48 h APF. (A) Uitlijnen van poppen op dubbelstokband. (B) Verwijdering van poppen uit de poppenkast door de pop voorste opening, het dorsaal snijden van de kast (B’) en het uittrekken van de pop (B”). Cirkelsymbolen vertegenwoordigden hetzelfde als figuur 2. (C) Overdracht van poppen naar…

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