Schiefelbein Lab Protocol for Root Cell
Protoplasting/Sorting/RNA Extraction for Microarray Analysis
November 2007
Growing Plants
Begin approximately 7-8 days before the cell-sorting date.
[Note: It is best to do biological replicates on different days, to account for environmental and experimental variation.]
- 1. Measure approximately 100 ul of seeds into an eppendorf tube.
[Note: Approximately 100 ul of seed is sufficient for lines carrying a very strong GFP expressing construct, such as the WER::GFP construct. Other (weaker) GFP lines will require more seed...approximately 2-5X more.]
- 2. Sterilize with 30% bleach 0.1% Triton X-100 for 12-15 minutes on rocker.
- 3. Remove bleach solution and rinse at least 4 times with sterile, double-distilled water.
- 4. Store seeds (in water) at 4C for 2-3 days.
- 5. Plate seeds densely onto plates containing MS Growth Media (see formula below), in 2 rows per plate. (Approximately 4 plates will be needed.)
- 6. Incubate plates vertically under continuous light at 22C for approximately 4-5 days.
MS Growth Media (1 liter)
0.6 % agarose (6 grams/liter)
0.2 grams MES (sigma M-2933)
1% sucrose (10 grams/liter)
4.33 g of MS salts (Cat# 11117-066, Invitrogen)
pH to 5.7-5.8 with KOH
Cut screens for growing plants (Nitex 03-100/47 mesh (Sefar,
www.sefaramerica.com or 800-283-8182). They need to be cut slightly smaller than a square plate...approximately 3 inches X 3 inches (size = a standard size square PostIt note). Wrap 5 screens in foil; lay flat on autoclave with open end down. Autoclave: 45 mins sterilize, 20 mins dry. Roots cannot grow through the 100gauge mesh. When plating, lay the seeds in two neat rows. Use a transfer pipette to "paint" them down in a line. About 500 to 1,000 seeds per row.
Protoplasting Protocol
Roots will be ready to harvest after approximately 4-5 days of plant growth, when the roots are approximately 2-3 cm long. Begin cutting the roots approximately 100 minutes before the scheduled time for the cell sorting.
- 1. About two hours prior to the cell-sorting reservation time, get wet ice and label 2 tubes [BD Falcon 5 ml polystyrene round-bottom tube, ref 352054 or 352058, this tube fits the cell-sorter the best] per sample:
- One tube is for the protoplasts, write "your sample name" and "cells" on the tube, leave it at room temperature until protoplasts are ready.
- The second tube is for the cell sorting technician to collect the sorted cells. Label this one: "collection", "the sample name" and "sorting date", put 450 ul RLT buffer and 4.5 ul beta-mercaptol ethanol into this tube and leave on wet ice.
[Note: This is the only step that uses ice...everything else is done at room temperature.]
- 2. Thaw 15-ml tube of enzyme (Solution B) and 1 tube of solution A 15 min before the digestion start time. [Note: Solutions A and B may be made fresh or may be made at an earlier time and frozen.]
- 3. Open the plates of seedlings and decant all the water and recap the plates. (Keep the plates in a vertical orientation as much as possible.)
- 4. Slice the roots from the bottom using a scalpel blade (about 1-2 mm length...approximately 1/3 of the end of the roots), chop the roots up just a little, four or five strokes with a sterile blade. After slicing all the roots from the plates of a particular group, scrape all the roots into a dish with solution B (enzyme solution). Gently mix the roots so that they are all in contact with the solution.
[Note: The dish is a 5cm-diameter disposable Petri plate. Place a 70 um strainer [BD Falcon #352350] in the dish and then pour about 5 ml solution B into it. (About 1 ml solution B is needed per plate of roots.)]
- 5. Place the dish on a rotating platform and allow to rotate at 85 rpm for one hour. At two times during this one hour period (after 20 min and after 50 min), gently mix the roots with the tip of a transfer pipet and lift the strainers partly out of the Solution B.
- 6. After one hour, gently mix the roots again and then lift the strainer out of the solution.
- 7. Place a small amount of the root material (that remains on the strainer) into a 15 ml tube (to help make a pellet). Gently aspirate liquid from outside of the strainer (using a transfer pipette); discard the strainer; and aspirate all the liquid from the Petri dish into the 15 ml tube (tilt the Petri dish to help).
- 8. Centrifuge the 15 ml tubes at 200g (1000 rpm) at 22 C for 6 min in a tabletop swinging bucket centrifuge. Remove tubes from centrifuge immediately after spinning.
[While waiting for spinning, set up the filtering: two 50 ml tubes; 70 um-white strainer sitting on the left tube and 40 um-blue strainer sitting on the right tube.]
- 9. Aspirate the supernatant and discard it. (Can aspirate faster at the beginning and slow down when getting close to the pellet.) Do not disturb the pellet.
- 10. Add 450 ul of Solution A to pellet and resuspend pellet carefully using a P1000 blue tip that is cut off at end. Mixture should appear homogeneous with no clumps of tissue.
- 11. Transfer Solution A + cells through the 70 um strainer (can tap the tube to aid filtration. Use another 450 ul of Solution A to wash the centrifuge tube and the 70 um strainer once.
- 12. Lift the 70 um strainer; Aspirate the liquid (using a 1 ml pipettor) from the outside of the 70 um strainer; pass all liquid through the 40 um strainer. DO NOT FORCE TO STRAIN...allow gravity to do the job.
- 13. Lift the 40 um strainer, aspirate the liquid from the outside of the 40 um strainer, combine all the liquid into a 5 ml polystyrene round bottom tube.
Take this tube, the labeled collection tube, and a container of dry ice to the FACS Facility for the protoplast sorting.
Preparing Protoplast Solutions
(Fisher and Sigma catalog numbers are listed but any standard reagents can be used for Soln A.)
For the other items, use the reagents listed.
Solution A. (This needs to be made fresh each time.)
Need pH 5.5, using 1 M TRIS, carefully, add ul quantities; it reaches pH quickly, e.g., < 100 ul.
600 mM Mannitol
2mM MgCl2
0.1% BSA
2 mM CaCl2
2 mM MES
10 mM KCl
For 200 ml
21.86 g (Sigma M-4125)
80 mg (Fisher C79-500)
0.2 g (Sigma A-3912)
59 mg (Fisher M33-500)
78 mg (Sigma M-2933)
150 mg (Sigma P-3911)
Solution B
Aliquot 50 ml of Soln A and add:
0.75 g cellulysin (Calbiochem, cat no. 219466)
0.05 g pectolyase (Sigma, cat no. 3026)
(50 ml of soln B is enough for 10,000 arabidopsis roots that are about 1 week old)
Cell Sorting:
After generating the protoplasts, they must be immediately taken to the Flow Cytometry Facility and sorted as soon as possible. The cells are to be collected in a 5 ml tube (containing 450 ul RLT + 4.5 ul BME) on ice. After collection, the tubes should be frozen by immediately placing them on dry ice. The tubes of collected cells are stored at -80C in this solution until they are used for RNA extraction.
Figure to the right shows an example of sorted cells.
Total RNA Extraction:
Using the Qiagen Rneasy Micro Kit
(Carry out the procedure at room temperature without disruption)
- 1. Thaw the frozen solution of RLT (including β-ME) + sorted cells. (This will be approximately 500-600 ul.)
[Note: If the number of sorted cells is less than 5000, add 20 ng carrier RNA. Generally, this is not necessary with WER::GFP sorted roots.]
- 2. Remove 350 ul of the RLT + sorted cells and place into new 1.5 ml Eppendorf tube.
- 3. Add 1 volume (350 ul) of 70% ethanol. Mix well by pipetting.
- 4. Apply the solution to the RNeasy MinElute Spin Column in a 2 ml collection tube.
- 5. Spin at 10,000 rpm in a microcentrifuge for 15 seconds. Discard the flow-through solution.
- 6. Add 700μl of Buffer RW1. Spin at 10,000 rpm for 15 sec. Discard the tube and the flow-through solution.
- 7. Transfer the RNeasy MinElute Spin Column into a new 2 ml collection tube.
- 8. Add 500μl of Buffer RPE onto the column. Spin at 10,000rpm for 15 seconds. Discard the flow-through solution.
- 9. Add 500μl of 80% ethanol (room temperature) to the column. Spin at 10,000g for 2 minutes. Discard the tube and the flow-through solution. (Do not let column touch the flow-through solution.)
- 10. Transfer the RNeasy MinElute Spin Column into a new 2 ml collection tube.
- 11. Open the cap of the spin column. Centrifuge at 12,000 rpm for 5 min with open caps. Discard the tube and the flow-through solution.
- 12. Transfer the RNeasy MinElute Spin Column to a new 1.5 ml collection tube.
- 13. Pipet 14 ul of RNase-free water directly onto the center of the silica-gel membrane. Close the tube gently, and spin at 12,000 rpm for 1 minute. This step elutes the RNA into a volume of approximately 12 ul.
- 14. Transfer 2 ul. of the RNA into a new tube. Label all of the tubes with an unambiguous number and a name. [For each RNA sample, there will be a 2 ul aliquot (used for testing RNA quantity/quality) and a 10 ul aliquot (used for microarray).
- 15. Store RNA samples at -80C.
Note: If the RNA will be used for other purposes (e.g. RT-PCR), then DNase treatment is recommended. (This is done after the RW1 buffer step.)
Note: For microarray analysis of whole roots (unsorted cells), the Qiagen RNeasy protocol can be used directly with root tissue to obtain total RNA. In this case, homogenization of the cells/tissue is necessary and the QIAshredder is used.
Microarray Analysis
The RNA obtained from this method is suitable for generating labeled nucleic acid for microarray hybridization or other downstream applications.
Reference:
Birnbaum et al. (2005) Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines.
Nat Methods 2:615-619.
Processing Gene Expression Data
Samples from various knockout specimens were tested on the Affymetrix ATH1 Genome array to
obtain the mRNA expression of genes [1]. The .CEL files so obtained from the experiment were preprocessed using the Robust Multichip Average (RMA) method, via the R
programming language package affy [2].
As mentioned here [3], the Affymetrix chip description file (CDF) has some problems associated with probe definitions and cross-hybridization. Therefore, a custom CDF from Brainarray Microarray Lab at the University of Michigan was used. This custom CDF uses
The Arabidopsis Information Resource (TAIR) annotation [4] and was implemented via
the Bioconductor package in R [5]. Using this annotation, the dataset has 21025 genes. The R code used to perform the preprocessing of
.CEL file is provided [6].
The TAIR website [7] provides various gene, process and pathway annotation files which allow us to map the TAIR IDs to gene names, GO processes etc. It also provides a nice interface to look up information about genes of interest.
