Source: Kao, S. Y., et al. Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches. J. Vis. Exp. (2019).
The Drosophila flight muscles are used as a model system for muscle development and physiology. This video describes the flight musculature of adults and highlights a protocol to dissect developing indirect flight muscles that are suitable for RNA sequencing from pupae.
This protocol is an excerpt from Kao et al., Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches, J. Vis. Exp. (2019).
1. IFM Dissection After 48 h APF
2. Pellet and Preserve the IFM Sample
Figure 1: Dissection of IFMs after 48 h APF. (A) Aligning of pupae on double-stick tape. (B) Removal of pupae from the pupal case by opening anteriorly, cutting the case dorsally (B'), and lifting out the pupa (B''). Circle symbols represented the same as Figure 2. (C) Transfer of pupae to buffer. (D) Removal of the abdomen by cutting with scissors (yellow double arrows) and separation from thoraxes (D'). (E,F) Addition of clean buffer (E), then cutting of thoraxes in half longitudinally (F,F'). (G,H) Dissections can be performed under white light (G) or fluorescence to visualize the GFP (H); cutting of the IFMs on one side (G'), then the other side (G''); lifting out of the thorax with forceps (outlined in grey) (G'''). (I,J,K) Collection of IFMs in buffer (I) and removal of contaminating ventral nerve cord (VNC), gut, and jump muscle (TDT) (J) to generate a clean IFM sample (K). TDT has lower GFP expression and a different shape than IFM fibers (J'', K'). (L,M) Use of forceps to transfer IFMs (L) to a microcentrifuge tube (M). Scale bars = 1 cm (A,E,M), 1 mm (B-D',F-L). Please click here to view a larger version of this figure.
Figure 2: IFM preservation and RNA isolation details. (A) IFMs are pelleted by centrifugation for 5 min at 2000 x g. (B) IFM pellet (arrow) and pellet under fluorescence (B'). (C) Removal of all buffer with a pipette tip. (D) For RNA extraction, resuspension of pellet in isolation buffer. This step can be skipped to dry-freeze dissected IFMs. (E) Freezing of sample in liquid nitrogen or on dry ice and storage at -80 °C. Scale bars = 10 cm (A), 1 mm (B,B'), 1 cm (C,D,E). (F) Nanograms (ng) of total RNA from dissected IFM obtained per fly at 16 h APF, 24 h APF, 30 h APF, 48 h APF, 72 h APF, 90 h APF, and 1 d adult. Error bars = SD. (G) Total RNA isolated from IFM dissected from 50 1 d adult flies using different extraction methods. Error bars = SD. (H) Representative traces to assay RNA integrity after different extraction methods. The ribosomal bands run just below 2000 nucleotides (nt) and the marker band at 25 nt. Additional traces available in Supplemental Figure 1. (I) Representative traces of a freshly isolated RNA sample (top), a sample freeze-thawed 25x on dry ice (second plot), a sample left for 4 h on the bench (third plot), and a sample treated with RNase A (bottom plot). Note complete degradation of RNA upon addition of RNase A. (J) RT-PCR gel from kits as labeled for bru1 and rp49. The relative intensity of the bru1 band normalized against rp49 is plotted below. Error bars = SEM (unpaired t-test, p = 0.0119). Please click here to view a larger version of this figure.
Supplemental Figure 1. (A,B,C) RNA yields from samples of the same genotype dissected by the same researcher in the same week. After all samples were dissected, RNA was isolated and measured the same day. (A) Nanograms (ng) of total RNA obtained from IFM dissections per 1 d adult fly. Error bars = SEM. (B) Total RNA obtained from dissected IFM per fly at 30 h APF, 48 h APF, 72 h APF and 1 d adult. (C) Total RNA isolated from IFM dissected from 50 1 d adult flies using different extraction methods. (D) Total RNA concentrations per fly from dissected legs, jump muscle (TDT) and IFM. More RNA is obtained from the larger IFMs. Error bars = SD. (E) Total RNA concentrations per fly of IFM dissected from controls compared to RNAi or mutant samples at 30 h APF, 72 h APF and 1 d adult. For mutants, w1118 was used as wildtype control. Mutant data are compiled from bru1-IR, salm-/- and another RNA-binding protein mutant. Note that for these manipulations, RNA yields are decreased in 1 d adult due to muscle atrophy and loss, so more flies need to be dissected to obtain sufficient quantities for omics approaches. Errors bars = SD. (F) Additional traces showing RNA integrity for the RNA isolation methods shown in Figure 2G and in C. Please click here to view a larger version of this figure.
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 |