Drosophila RNAi Cell Microarray Methods

Table of Contents

  1. Introduction
  2. dsRNA Preparation
  3. Microarray Printing
  4. Cell Lines and Culture Technique
  5. Seeding of Arrays
  6. Fixation of arrays, Immunofluorescence, DNA, and Actin Staining
  7. Image Acquisition and Analysis
  8. Useful Links
  9. Contact Information
  10. Acknowledgements
  11. Sabatini Lab Website
  12. The Whitehead Institute for Biomedical Research
1. Introduction

Welcome to the Drosophila RNAi Cell Microarray Home Page. This website describes a technique for rapid microarray-based analysis of RNA interference (RNAi) induced phenotypes in cultured Drosophila cells and is meant to accompany the paper "RNAi living cell microarrays for loss-of-function screens in Drosophila melanogaster cells." Double stranded RNAs (dsRNA) that target specific genes are printed onto a glass microarray slide using a commercially available microarrayer. Adherent, phagocytic Drosophila cells (Kc167, S2R+) are then cultured on the arrays. The result is a living microarray which consists of a lawn of cells. Cells that land on the dsRNA spots exhibit localized phenotypes in response to the knockdown of the gene targeted by the dsRNA. The image to the right is a simple example of this method. In the image, nuclear (left) and phospho-dAkt staining (right) were captured with a 5x objective. The black spots in the upper part of the left panel are areas devoid of cells because of the localized knockdown of Drosophila Inhibitor of Apoptosis 1 (DIAP1). In addition, the dsRNA targeted to dPTEN (Phosphatase and Tensin Homologue) leads to the hyperphosphorylation of dAkt. Increased immunofluorescent staining for P-dAkt can be seen in the middle row of the right panel. The rings in the bottom of each image denote the location of dsRNA targeted to GFP, that served as a negative control.

Success of this technique depends upon several important factors. This site provides detailed instruction on how to successfully create RNAi cell based microarrays. Each section of this page will describe a particular aspect of the RNAi cell microarray process. Sections will begin with an introduction of the topic at hand, followed by a materials list, methods, and end with some notes.

 

2. dsRNA Preparation

Introduction

Fastidious design, synthesis and purification of dsRNA will increase the quality of RNAi microarrays. While each laboratory may have their own methods of dsRNA preparation, we have tested several methods and found that steps presented below work best. We describe a protocol that details dsRNA primer design, RT-PCR, in vitro transcription and purification. This protocol can be readily adapted for high-throughput synthesis of dsRNA in a 96 well plate format. Click here for a flow-chart protocol of 96 well plate based IVT.

Materials for dsRNA Preparation
Item
Product Number, Supplier
Utility
http://www.flybase.org
Obtain cDNA sequence for targeted gene
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi
Designs primers for dsRNA
Qiagen RNeasy Mini Kit
74103, Qiagen
Purify total mRNA from Drosophila cells
Qiagen OneStep RT-PCR Kit
210210, Qiagen
Amplification of PCR products from total mRNA
MEGAscript Transcription Kit (Bulk)
B1334, Ambion
In vitro transcription of dsRNA from PCR product
Nuclease Free Water
9332, Ambion
Various
1 M Tris Buffer (pH 7.0)
9851, Ambion
Used to prepare 10 mM Tris buffer
DNAse I
2224 (bulk), 2222 (individual), Ambion
Digestion of PCR template
96 well round bottom plate
29445-112, VWR
IVT reaction plate
96 well V bottom plate
29444-400, VWR
dsRNA storage
PCR Fragment Vacuum Purification Plate
MANU03050, Millipore
Vacuum purification of dsRNA
Vacuum Manifold for PCR Purification Plate
MAVM0960R, Millipore
Vacuum purification of dsRNA

Protocol

Primer Design

  1. Obtain cDNA sequence(s) for target gene(s) from Flybase.
  2. Use Primer3 and cDNA sequence to formulate primers for fragment amplification. Primers are typically designed to generate 700-800 bp length PCR products.
  3. Add the T7 RNA Polymerase binding site (5’-GAATTAATACGACTCACTATAGGGAGA-3’) to the 5' end of each primer.
  4. Order or synthesize primers.

Total mRNA Purification

  1. Total mRNA is purified from Drosophila S2 cells using the Qiagen RNeasy Mini Kit. See kit for protocol.

RT-PCR

  1. Carry out RT-PCR reactions with primers and total mRNA (template) generated above using a commercially available RT-PCR kit (e.g. Qiagen One Step RT-PCR kit).
  2. Purification of DNA fragments is not necessary.

In Vitro Transcription (IVT) (Click here for a flow chart protocol of IVTs in a 96 well format)

  1. Add 5 ul of each template DNA to a 20 ul (final volume) in-vitro transcription reaction as described by the Ambion MEGAscript instructions.
  2. Incubate IVT reactions overnight (~12 hours) at 37°C.

Vacuum Purification of dsRNA

  1. Add 2 U of DNAse I in 80 ul of nuclease free water to IVT reaction, mix and incubate for 0.5-1.0 hour at room temperature.
  2. Set up Millipore PCR vacuum purification filter plate and manifold. Set vacuum pressure to 5 psi and load one sample into each well. Vacuum purify samples for 45 minutes at 5 psi.
  3. Remove purification plate from vacuum manifold.
  4. Add 50 ul of 10 mM Tris pH 7.0 Buffer to each well.
  5. Seal plate with adhesive foil and shake on a plate shaker for 20 minutes (avoid vigorous shaking as these plates CANNOT be centrifuged).
  6. Add another 50 ul of 10 mM Tris pH 7.0 Buffer to each well, mix 5x and transfer sample to storage plate or eppendorf tube.
  7. Quantify dsRNA using spectrophotometer with a 10 mM Tris pH 7.0 Buffer blank.
  8. Store dsRNA at -20°C until use.

Preparation of dsRNA for Printing: We print dsRNA in a solution of 10 mM Tris pH 7.0 Buffer with 500 mM NaCl. The normal volume of the sample that we print is 20 ul, with a dsRNA concentration of 0.05 to 0.4 ug/ul.

  1. Prepare a master mix of 1.25 M NaCl in 10 mM Tris Buffer.
  2. Aliquot 8 ul of master mix into a tube or a plate well that will contain the dsRNA.
  3. Add an appropriate amount of dsRNA.
  4. Bring volume to 20 ul with 10 mM Tris pH 7.0 Buffer.
  5. Transfer sample to 384 well plate for printing.

NOTES

3. Microarray Printing

Introduction

We printing the dsRNA using a commercially available microarrayer. As arraying techniques and protocols are fairly standard, arraying specifications and a list of the materials utilized is provided here along with some helpful tips to successfully print RNAi microarrays.

Materials for Microarray Printing
Item
Product Number, Supplier
Utility
Polypropylene 384 Well Plate
EK-30202, E&K Scientific
Storage of dsRNA
Robotic Microarrayer
PixSys 5500A, Cartesian Technologies
Printing of microarrays
Stealth Pins
ArrayIt SMP7, Telechem

Delivery of dsRNA to microarray slide

Micro Cleaning Solution
Pin Cleaning
GAPS Slides
40015, Corning
Preprint Substrate
DS8 Custom Slides
C100-5973-M20, Erie Scientific

Slides with proprietary cell attachment chemistry

Rhodamine B
R-6626, Sigma
Printed on arrays as fluorescent reference point
poly-d, l- lactic acid (PDLA)
16585, Polysciences
Polymer that sequesters Rhodamine B
Methyl Salicylate
M-2047, Sigma
Solvent for PDLA and Rhodamine B

Before Printing:

  1. Clean Pins: Prior to printing, the pins used to print the arrays should be cleaned, washed, and dried. The pins are cleaned by sonication in the Micro Cleaning Solution from Telechem for 30 minutes, washing with distilled water and air drying.
  2. Program Arrayer: The arrayer is programmed to deposit the dsRNA onto the glass slides in the desired pattern.
  3. Check Arrayer: Ensure that the arrayer parts (vacuum, wash basin, humidity, etc.) are functioning correctly.
  4. Place Slides in Arrayer: Position slides on the arrayer and make any other final preparations for the print.
  5. Other: The specifications listed here result in optimal dsRNA arrays:
Arrayer Specifications
Pins
SMP7
Spotting Time
25 ms
Preprint Substrate
GAPS slides
# of Pre-Print Spots
12
Relative Humidity
70%
Print Substrate
Erie DS8 Slides

Printing Substrate: We initially developed a system of coating VWR glass microscope slides in a 0.25 mg/ml solution of Concanavalin A (see here for coating protocol). Concanavalin A is a lectin that can bind glycosyl and mannosyl groups which are attached to extracellular proteins. By this virtue, cells are able to adhere more vigorously to surfaces that are coated with Concanavalin A. To avoid inconsistencies inherent in this "home-made" method, we collaborated with Erie Scientific to formulate a cell attachment chemistry that would be suitable for our microarray method resulting in a slide coat called DS8. We use slides prepared by Erie Scientific with this coating. Erie prepares typical flat-glass slides or etched surface (ES) slides with this coating.

Spot Spacing and Spot Size: While the typical spot spacing rule of thumb for cDNA arrays is to allow spot to spot spacing of 1.5 times the diameter of each spot, more space should be allowed with cell microarrays. Smaller spots (such as those printed on cDNA arrays) are not as uniform, nor do they allow enough cells to be statistically significant when doing high-content analysis. Using an smp7 pin, spot sizes of ~235 um are achievable resulting in phenotypic spots of ~225 um. Spot to spot distance is kept around 500 um, allowing for about 5000 spots on one standard microscope slide.

"Marker" Spots: In order for each individual spot to be photographed at high magnification, the arrays are printed with "marker" spots in defined locations. A polymer of poly d,l-lactic acid (100 mg ml-1) in methyl salicylate with 4 uM rhodamine B dye (Protocol here) acts as a fluorescent marker and is printed a set distance away from the corners of the arrays. Alternatively, DIAP1 dsRNA spots can serve as markers because of the highly penetrant of the cell death phenotype induced.

 

4. Cell Lines and Culture Techniques

Introduction

The best Drosophila cell lines for use on RNAi microarrays tend to be adherent, phagocytic cell types. We obtained cells from frozen stocks from the Perrimon Laboratory at Harvard Medical School, which also maintains the Drosophila RNAi Screening Center (DRSC). The DRSC website is an invaluable resource for information pertaining to the culturing of Drosophila cells and the utility of RNAi in fly cells. The protocol that follows has been optimized for use of Drosophila cells on RNAi microarrays.

Materials for Cell Culture
Item
Product Number, Supplier
Utility
Schneider's Drosophila Medium
11720-034, Gibco
Propagation of Drosophila cell lines
Inactivated Fetal Calf Serum
n/a
Media supplement
Penicillin and Streptomycin
n/a
Media supplement
T75 Cell Culture Flasks
430198, Corning
Propagation of Drosophila cell lines
Cell Scrapers
3010, Corning
Cell Harvesting
Custom made incubator
Humidity controlled cell incubator

Cell Culture Protocol

Medium Preparation

  1. Bring 500 mL of Schneider's Insect Medium to room temperature. Also thaw inactivated fetal calf serum (IFCS), Penicillin, and Streptomycin.
  2. Add 55 mL of IFCS to medium (final IFCS concentration of 10%).
  3. Add 25,000 units of Streptomycin and 25 mg of Penicillin to medium.
  4. Store medium at 4°C in dark when not in use.

Culturing Cells

  1. See chart below for recommended splitting frequencies. Perform all culturing of cells in a sterile environment.
  2. Scrape cells off of flask surface. Avoid formation of bubbles in medium.
  3. Pipet cells up and down 10x at medium speed with a 10 mL pipet.
  4. Deposit cells in a 50 mL conical tube.
  5. Count cells and determine cell density.
  6. Aliquot desired number of cells into a new 50 mL conical. Add an appropriate amount of new medium such that each new culture flask will have 12 mL of medium.
  7. Deposit 12 mL of resuspended cells into new culture flasks.
  8. Passage cells again in 3-4 days. Monitor cultures for health while they are growing.
Recommended Cell Splitting Regimes ("M" = 10e6)
Cell Type
Split Frequency
Initial Culture Density
Kc167
Every 4 days
80 M cells in 12 mL
S2R+
Every 4 days
15 M cells in 12 mL

Notes

5. Seeding of Arrays

Introduction

Throughout this protocol, the addition of cells to the arrays is referred to as "seeding" the arrays. Cells can be starved on the arrays or simply grown to a thick density and starved prior to seeding. We have found that using cells that have grown for 96 hours gives highly reproducible results on the arrays.

Materials for Processing Arrays
Item
Product Number, Supplier
Utility
Lab-Tek Square Petri Dishes
25387-024, VWR
Growth of arrays
Coulter Z2 Particle Count & Size Analyzer
Z2, Beckman Coulter
Cell Counting
Serum Free Insect Medium
10797-025, Gibco
Starvation of phagocytic cells
Schneider's Drosophila Medium
11720-034, Gibco
Propagation of Drosophila cells

Array Seeding Protocol

Cells: Growth Times

  1. Seed arrays with cells that have been growing for ~96 hours. 1 hour prior to seeding, remove the medium that is on the cells and replace with 10 mL of Serum Free Medium. Incubate for one hour and then harvest cells. This starvation step is meant to ensure that the cells will be starved.

Harvest Cells

  1. Harvest cells via scraping. Pipet cells up and down 10x in a 10 mL pipet and transfer to a 50 mL conical.
  2. Count cells using preferred cell-counting method.
  3. Aliquot the appropriate amount of cells into a 15 mL conicle. Use between 10e6 and 25e6 cells for one square petri dish. Standard seeding density for a 3 day experiment is 15e6 cells per dish.
  4. Centrifuge cells at 1000 rpm for 5 minutes.

Resuspend Cells

  1. Aspirate supernatant off of pelleted cells.
  2. Resuspend pellet in 1 mL of regular Drosophila medium, pipet up and down 10x.
  3. Transfer cells to a 50 mL conicle containing 24 mL of medium.
  4. Pipet cells up and down 10x in a 10 mL pipet to ensure that the cells are in a single cell suspension.

Seed Arrays

  1. Arrange arrays in desired configuration in square plastic petri dish. If you are only seeding 2 arrays, include a third "space filling" microscope slide.
  2. Add cells to the right-most side of the arrays with a 25 mL pipet. Add cells at a medium pace, such that the medium moves across the slides without stopping.
  3. Gently rock the petri dish from side to side to ensure proper cell distribution.

Incubate Arrays

  1. Allow arrays to grow for 3-4 days. Cell number may be modified for shorter or longer incubation times. Monitor arrays during growth to ensure proper cell attachment and RNAi effects.

Notes

 

6. Fixation and Staining of Arrays

Introduction

Described below are the procedures following cell growth on the arrays. This usually consists of fixing the arrays, permeabilizing and probing them with primary and secondary antibodies, staining for nuclei and actin, and mounting the arrays with glass coverslips. If antibody or actin staining is not desired, permeabilization may be removed from the protocol.

When this protocol calls for humidified petri dishes, we are referring to square dishes that contain a sheet of moist whatman paper. Furthermore, all rinses should be performed in coplin jars containing PBS+ (Note: The presence of Ca++ and Mg++ ions vastly improved the staining of F-actin with FITC conjugated fluorescein.).

Materials for Processing of Arrays
Item
Product Number, Supplier
Utility
General
Coplin Jars
25457-006, VWR
Rinsing and staining of arrays
Phosphate Buffered Saline (with 1 mM MgCl2, 1 mM CaCl2) (aka PBS+)
n/a
Rinsing
Lab-Tek Square Petri Dishes
25387-024, VWR
Staining of arrays
Fixation
Paraformaldehyde
158127, Sigma
Fixation of cells
Permeabilization
Triton X-100
789-704, Roche
Permeabilization of samples
Immunofluorescence, Actin and DNA Staining
BSA
A9418, Sigma
Antibody blocking and incubating solution
Normal Donkey Serum
Antibody blocking solution
Phospho-dAkt Primary Antibody
4054, Cell Signalling
Probing for hyper phosphorylated dAkt
Cy3 labeled anti-rabbit secondary antibody
Probing for hyper phosphorylated dAkt
Fluorescein conjugated phalloidin
F-432, Molecular Probes
F-actin staining
Hoechst 33342 dye
H-3570, Molecular Probes
DNA staining
Mounting of Slides
Vectashield Mounting Medium
H-1000, Vector Laboratories
Mounting of microarrays
26 X 60 mm Cover Glasses
48393-106, VWR
Mounting of microarrays
Shiny Top Coat Nail Polish
23135-90-00, Sally Hansen
Mounting of microarrays

Fixation and Staining Protocol

Fixation: Upon the termination of an experiment we perform fixation as follows:

  1. Arrays are rinsed with PBS+ in coplin jars then placed in humidified square petri dishes (See Image 1).
  2. Add 600 uL of fixative (3.7% paraformaldehyde, 4.0% sucrose in PBS+) to the lower right corner of the slide and allow it to cover the whole area of the slide (See Image 2) (note: To uniformly distribute the fixative on the array, gently tilt the petri dish).
  3. Incubate arrays at room temperature for 20 min.
  4. Tip the petri dish such that the paraformaldehyde pools in the lower right corner and aspirate (See Image 3).
  5. Using tweezers, remove arrays and rinse once in fresh PBS+.

Permeabilization: Proceed directly to nuclear staining if no other staining procedures are being followed.

  1. Using tweezers, place freshly fixed and rinsed array in a coplin jar containing 0.1% Triton X-100 (in PBS+).
  2. Incubate at room temperature for 30 minutes.
  3. With tweezers, remove the arrays and rinse once in fresh PBS+. Return arrays to humidified petri dish.
  4. To ensure that the arrays do not dry, make sure the next reagent is added quickly after the slides are placed in the humidified petri dish.

Blocking and Immunofluorescent Staining

    1. In a humidified petri dish, add 600 ul of blocking solution (1.0% BSA, 1:100 NDS in PBS+) to arrays as before and incubate for 1 hour at room temperature.
    2. Aspirate blocking solution as before and add 1:500 dilution of primary antibody in antibody incubating solution (0.5% BSA in PBS+). Gently tilt petri dish to ensure even distribution of antibody solution. Incubate for 1 hour at room temperature or overnight at 4°C (This will vary depending on the primary antibody and its specificity. For example, for P-dAkt staining, we found that overnight incubation resulted in a more even staining of the arrays).
    3. After incubation with primary antibody, aspirate antibody and rinse arrays in PBS+ as before.
    4. Return arrays to humidified dish and add 600 ul of blocking solution. Incubate at room temperature for 20 minutes.
    5. Aspirate arrays and add 600 ul of secondary antibody solution (1:250 secondary antibody in antibody incubating solution). If F-actin staining is desired, include FITC-phalloidin (also a 1:250 dilution) along with secondary antibody. Incubate in the dark for 40 minutes at room temperature.
    6. When incubation with secondary antibody is complete, rinse arrays in PBS+, then dry the back and sides of arrays with a paper towel. Proceed with nuclear staining.

Nuclear Staining

    1. While arrays are either fixing or staining with secondary antibody, prepare nuclear staining solution as follows: Add 50 mL PBS+ to a coplin jar that has been wrapped in aluminum foil. To this coplin, add 5 ul of Hoechst 33342 (10 mg/ml by supplier) or preferred nuclear dye. Mix contents of jar by inversion.
    2. Rinse arrays in PBS+ and place in foil-covered coplin jar. Incubate at room temperature for 20 minutes.
    3. Remove arrays, rinse in PBS+, dry back and sides of arrays with paper towel, and lay arrays down on a paper towel on benchtop for mounting.

Mounting of Arrays

    1. To the left edge of the array, add a column of drops of VectaShield mounting media (note: home made PPD based media can also be used, but VectaShields lower viscosity greatly diminishes the occurrence of air bubbles).
    2. Place the lower left corner of the coverglass onto the lower left corner of the array, and lower the left edge of the coverglass such that the mounting medium forms a "front" on the left edge of the array. Without pause, gently (but not slowly) lower the right side of the coverglass onto the array as the "front" of the medium advances across the arrays. If bubbles seem imminent, you may use your pinky finger to "massage" the top of the coverglass and force the bubbles beyond the moving front of mounting medium.
    3. Once the coverglass is on the array, aspirate excess media that bleeds out from under the glass. Be careful not to move the cover slip.
    4. Seal the coverglass to the microscope slide using Shiny Top Coat fingernail polish. There are several varieties of nail polish, and we have found that the Shiny Top Coat type works better than most at sealing arrays.
    5. Place arrays in a dry chamber (square petri dish with dry piece of whatman paper on bottom). Store arrays at 4°C in the dark until image acquisition.

 

7. Image Acquisition and Analysis

Introduction

In addition to large 5x images of the entire array area, 40x images of the cells on each particular spot are acquired using an automated fluorescent microscope. The computer program KS400 (Carl Zeiss) has a feature known as "mark and find" which allows for the acquisition of 40x images at each spot of dsRNA with various exposures. Spot locations are defined by the user via a position list that is utilized by KS. As this program was custom made by Zeiss, Zeiss may be contacted for more information.

Detailed analysis of images acquired by the "mark and find" method was performed using CellProfilerTM. CellProfilerTM is a multifunctional program that was designed to analyze large stacks of images. This capacity makes CellProfilerTM ideal for analyzing the large numbers of high content images with multiple exposures that are the result of a cell microarray experiment. CellProfilerTM will be available to the research community at large in the near future.

Materials for Image Acquisition and Analysis
Item
Product Number, Supplier
Utility
Automated Fluorescent Microscope
Axiovert 200M, Zeiss
Image acquisition
Imaging software suite
KS400 3.0, Zeiss
Image acquisition
CellProfilerTM
Analysis of high-content images

 

8. Useful Links

 

9. Contact Information

For questions, comments, or observations, contact:

David M. Sabatini, M.D., Ph.D. (Material requests or procedural information)

Douglas B. Wheeler (Website related questions)

Whitehead Institute for Biomedical Research

9 Cambrige Center

Cambridge, MA 02143

Phone: 617.258.6407 or 617.258.9480

Fax: 617.258.5123

10. Acknowledgements

The Sabatini lab would like to thank those who directly and indirectly contributed to this project. Thank you to Norbert Perrimon, Michael Boutros and Tom Volkert for helpful discussions.