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Focus: High-Throughput mRNA Isolation

Automated Poly(A)+ mRNA Isolation From Plant Tissues Using the PolyATtract^® System 1000

By Tracy Worzella, B.S. and Terri Grunst, B.S.
Promega Corporation

Introduction

The PolyATtract^® System 1000^(a) isolates mRNA directly from crude lysate, eliminating the need for total RNA isolation. The system uses Biotinylated Oligo(dT) and Streptavidin MagneSphere^® Paramagnetic Particles* to capture mRNA and is adaptable to automated formats. In these experiments we demonstrate the application of the PolyATtract^® System 1000 to a variety of plant tissue samples using three different automated formats: the Labsystems KingFisher™, Labsystems KingFisher™ mL and the Beckman Biomek^® 2000 (Figure 1). All of these automated formats will purify mRNA with little to no hands-on requirements from the user. The plant tissue mRNA purified using these automated procedures serves as a good template for RT-PCR analysis.

Instrumentation

The three different automated platforms used in this article possess different processing capabilities and require different levels of capital investment. The Beckman Biomek^® 2000 will process up to 96 samples in approximately an hour. This versatile, liquid handling instrument has multiple expansion options for handling many different applications.

The Labsystems KingFisher™ and KingFisher™ mL are economical magnetic handling instruments. The KingFisher™ can process up to 24 samples (two plates of 12 samples each) in approximately 15 minutes. Sample lysate volumes, however, are limited to 20µl. The KingFisher™ mL is a modified version of the standard KingFisher™ which processes sample volumes up to 1ml, but limits the number of samples to 15 per run. Because the KingFisher™ and KingFisher™ mL are magnetic handling instruments, applications are limited to processes using magnetic particles.

Preparation of Plant Lysates and Reagents

For this article, we processed leaf and stem tissue from alfalfa, Arabidopsis, tomato, corn seedling and tobacco as described below.

1. Crush 100mg of plant tissue with a mortar and pestle in the presence of liquid nitrogen.
2. Add 1.5ml of GTC Extraction Buffer to the plant tissue powder in the mortar after the liquid nitrogen has evaporated.
3. Further crush the powdered plant tissue in the buffer until plant tissue fibers can no longer be seen.
4. Remove the plant lysate to a new 1.5ml microcentrifuge tube and store on ice while preparing the remaining reagents.

Isolation of mRNA from different plant species and tissues may require optimizing the amount of tissue used to produce the lysate. The amount of tissue processed for these experiments provides a good starting point for optimizing the procedure. In these experiments, the Beckman Biomek^® 2000 and the Labsystems KingFisher^® instruments processed 0.3mg of plant tissue per well and the KingFisher^® mL instrument processed 10mg of plant tissue per tube. Each instrument has defined volume limitations. These experiments use the maximum volume that can be processed by each instrument, so any optimization of plant lysate preparation will need to remain within the volume processing capabilities of the specific instrument.

Reagent preparation consists of making hybridization solution and washing the Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) with 0.5X SSC. The volumes required for isolating mRNA in these automated formats depend on the instrument used and the number of samples being processed. Specific indications for each of the instruments are discussed in detail below.

Automated Isolation of Poly(A)+ mRNA Using the Beckman Biomek^® 2000

1. After sample lysate preparation, a master mix of hybridization solution is prepared by combining 7ml pre-warmed dilution buffer, 143.5µl b-Mercaptoethanol and 10.5µl of Biotinlyated Oligo(dT).
2. Preheat and leave the hybridization solution at 65^°C while preparing the rest of the reagents. The prepared hybridization solution is enough to process one 96 well plate of lysate samples on the Biomek^® 2000.
3. To prepare the SA-PMPs, wash 10ml (enough for 96 samples) of the SA-PMPs three times with 10ml 0.5X SSC.
4. After the wash steps, resuspend the SA-PMPs in 10ml 0.5X SSC.
5. The deck of the Biomek^® 2000 should be set to the initial deck configuration of the program as shown in Figure 2. Place the 65^°C hybridization buffer on the deck just prior to starting the run to minimize cooling of the buffer.
6. Place 20µl of prepared lysate in a 96 well V-bottom sample plate.
7. Place the sample plate on the deck of the Biomek^® 2000 instrument.
8. Once the deck of the Biomek^® 2000 has been set up, start the Biomek^® program (Table 1).

Automated Isolation of Poly(A)+ mRNA Using the Labsystems KingFisher™ Instrument

1. After sample lysate preparation, the lysate is stored on ice while preparing the remaining reagents.
2. A master mix of hybridization solution is prepared based on the number of samples to be processed. For each sample to be purified, combine 40µl of Dilution Buffer, 0.82µl b-Mercaptoethanol and 0.6µl Biotinylated Oligo(dT) Probe.
3. Preheat and leave the hybridization solution at 65^°C while preparing the rest of the reagents.
4. To prepare the SA-PMPs, wash 2ml (enough for 24 samples) of the SA-PMPs three times with 2ml 0.5X SSC.
5. After the wash steps, resuspend the SA-PMPs in 2ml 0.5X SSC.
6. After reagent preparation, the KingFisher™ plate is set according to Table 2 leaving the hybridization solution and SA-PMPs out of the initial plate setup. One column of the KingFisher™ plate is used per mRNA isolation.
7. After setting up the KingFisher™ plate, add 40µl of preheated hybridization solution to Row A containing the 20µl of lysate.
8. Immediately place the KingFisher™ plate into the KingFisher™ instrument and isolate mRNA using the program described in Table 3. The KingFisher™ beeps after the first mixing step, prompting the user to add 75µl of SA-PMPs to Row A.
9. Prepared mRNA is eluted into 100µl of water in Row E of the KingFisher™ plate.

Table 2. KingFisher™ Plate Setup.
Row A	20µl lysate and 40µl hybridization solution. Add 75µl SA-PMPs when prompted.
Row B	150µl 0.5X SSC
Row C	150µl 0.5X SSC
Row D	150µl 0.5X SSC
Row E	100µl water and 1µl RQ1 RNase-Free DNase(a) (Cat.# M6101)

 

Table 3. KingFisher™  Program for Isolation of Poly(A)+ mRNA.
Step	Process
1	The KingFisher™ instrument mixes the contents of row A for 2 minutes. The biotinylated Oligo(dT) anneals to the mRNA.
2	The instrument prompts the user to add SA-PMPs to Row A, then it mixes the contents of Row A for 2 minutes. The SA-PMP/mRNA complex is captured, then moved to Row B.
3	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Row C.
4	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Row D.
5	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Row E.
6	The instrument mixes the SA-PMP/mRNA complex to elute the mRNA in 100µl water. The SA-PMPs are captured and moved back to Row D for disposal.

Automated Isolation of Poly(A)+ mRNA Using the Labsystems KingFisher™ mL Instrument

1. After sample lysate preparation, the lysate is stored on ice while preparing the remaining reagents.
2. A master mix of hybridization solution is prepared based on the number of samples to be processed. For each purification, combine 400µl of dilution buffer, 8.2µl b-Mercaptoethanol and 0.6µl Biotinylated Oligo(dT) Probe.
3. Preheat and leave the hybridization solution at 65^°C while preparing the rest of the reagents.
4. To prepare the SA-PMPs, wash 8ml of the SA-PMPs (enough for 15 samples) three times with 8ml 0.5X SSC. After the wash steps, resuspend the SA-PMPs in 8ml 0.5X SSC.
5. After reagent preparation, the KingFisher™ mL strip tubes are set up according to Table 4 leaving the hybridization solution and SA-PMPs out of the initial strip-tube setup. One KingFisher™ mL strip tube is used per mRNA isolation.
6. After setting up the KingFisher™ mL strip tubes, add 400µl of preheated hybridization solution to Tube 1 containing the 750µl of lysate.
7. Immediately place the KingFisher™ mL strip tubes into the KingFisher™ mL instrument and isolate mRNA using the program described in Table 5.
8. The KingFisher™ mL beeps after the first mixing step, prompting the user to add 500µl of SA-PMPs to Tube 1.
9. Prepared mRNA is eluted into 100µl water in Tube 5.

Table 4. KingFisher™ mL Strip Tube Setup.
Tube 1	750µl lysate, 400µl hybridization solution. Add 500µl SA-PMPs when prompted.
Tube 2	500µl 0.5X SSC
Tube 3	500µl 0.5X SSC
Tube 4	500µl 0.5X SSC
Tube 5	100µl water and 1µl RQ1 RNase-Free DNase

 

Table 5. KingFisher™ mL Program for Isolation of Poly(A)+ mRNA.
Step	Process
1	The KingFisher™ mL instrument mixes the contents of Tube 1 for 2 minutes. The biotinylated Oligo(dT) anneals to the mRNA.
2	The instrument prompts the user to add SA-PMPs to Tube 1, then it mixes the contents for 2 minutes. The SA-PMP/mRNA complex is captured, then moved to Tube 2.
3	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Tube 3.
4	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Tube 4.
5	The SA-PMP/mRNA complex is washed in 0.5X SSC, captured and moved to Tube 5.
6	The instrument mixes the SA-PMP/mRNA complex to elute the mRNA in 100µl water. The SA-PMPs are captured and moved back to Tube 4 for disposal.

Analysis of Purified mRNA

Analysis of purified plant GAPDH mRNA by RT-PCR using the Access RT-PCR System^(a,b,c) (Cat.# A1260) is shown in Figure 3. Universal GAPDH primers were designed to amplify plant GAPDH regardless of template plant species. Known cDNA sequences for GAPDH from tomato (Acc# U97257), potato (Acc# U17005), zea mays (Acc# X07156), and bread wheat (Acc# AF251217) were compared at the nucleotide level. Primers were designed to regions 100% conserved at the amino acid level and greater than 80% conserved at the nucleotide level. The four most 3´ nucleotides of the primers are 100% conserved among all four known cDNA sequences. The expected PCR product size based on the known cDNA sequences is approximately 277bp.

Conclusions

We have demonstrated the isolation of mRNA from a variety of plant lysate samples on three different robotic instruments using the PolyATtract^® System 1000 (Cat.# Z5400, Z5420). The three instruments (Biomek^® 2000, KingFisher™ and KingFisher™ mL) used for this application represent different volume-handling and throughput processing capabilities. The PolyATtract^® System 1000 will purify mRNA with little or no hands-on requirements from the user on each of these platforms. We demonstrate the adaptability of the PolyATtract^® 1000 mRNA Isolation System to automated isolation formats, both liquid and nonliquid handling instrumentation. All samples processed in this automated format provide quality mRNA that is suitable for downstream applications such as RT-PCR. No cross-contamination was detected using these automated procedures (data not shown).

^(a)For Laboratory Use.

^(b)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

^(c)U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, which claim vectors encoding a portion of human placental ribonuclease inhibitor, are exclusively licensed to Promega Corporation.

*Products may be covered by pending or issued patents. Please visit our patent and trademark web page for more information.

MagnaBot, MagneSphere and PolyATtract are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.

Biomek is a registered trademark of Beckman Instruments, Inc. KingFisher is a trademark of Labsystems.

 

Figures for Automated Poly(A)+ mRNA Isolation From Plant Tissues Using the PolyATtract^® System 1000

Figure 1. Instrumentation used for the automated purification of mRNA with the PolyATtract® System 1000.

Figure 2. Initial deck configuration of the Biomek® 2000. The tools required include the Gripper and MP200, placed at position A1. A MagnaBot® 96 Magnetic Separation Device (Cat.# V8151) is located at position A2. Boxes of P250 tips are located at positions A3 and A4. A reservoir holder at position B1 holds a quarter reservoir with 10ml of prewashed SA-PMPs, and a vertically split quarter reservoir with 7ml of prewarmed hybridization solution and 12ml of Nuclease-Free Water. A second reservoir holder is placed at position B3 containing two quarter reservoirs: one reservoir for waste and one reservoir for 45ml of 0.5X SSC plus 1ml of RQ1 RNase-Free DNase (Cat.# M6101). A new 96 well V-bottom plate is placed at position B4. The V-bottom sample plate containing the prepared lysate is placed at position B2. Positions A5, A6, B5 and B6 remain empty.

Figure 3. Analysis of purified mRNA by RT-PCR. Prepared mRNA (5µl) was amplified using the Access RT-PCR System (Cat.# A1260) with the primers described. Reactions without reverse transcriptase were done as a control to assay for DNA contamination. Samples were placed in a licensed thermocycler running the following program: 45 minutes at 48°C, followed by 2 minutes at 95°C, followed by 40 cycles of: 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds finishing with a 2 minute incubation at 72°C. The RT-PCR samples (20µl each) were run on a 1.5% agarose gel. RT-PCR products were visualized by staining with ethidium bromide. Expected GAPDH RT-PCR products (~277bp) are seen from tobacco, corn (seedling), alfalfa, Arabidopsis and tomato leaf and stem tissues. Control reactions minus reverse transcriptase did not show any amplification products, indicating no DNA contamination (data not shown).

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