RLCS (Restriction Landmark cDNA Scanning) Protocol

RLCS (Restriction Landmark cDNA Scanning) Protocol for inmature barley grains (probably applicable for other plant materials)   RLCS that was developed by Suzuki et al. (1996) displays many cDNA species quantitatively and simultaneously at two dimensional gel spots ( RLCS profile ).   In this method, cDNA species of uniform length were prepared for each mRNA species using restriction enzymes.    After the restriction, enzyme sites were radiolabeled as landmarks, and then the labeled fragments were subjected to high resolution two-dimensional gel electrphoresis.   Since the location and intensities of spots reflect the expression of certain mRNA species, RLCS serves as a usuful cDNA display system that provides valuale information on expressed genes. References: 1) Suzuki, H. T. Yaoi, J. Kawai, A. Hara, G. Kuwajima and S. Watanabe, 1996.    Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis. Nucleic Acids Research 24(2): 289-294. 2) K. Mayumi, T. Yaoi, J. Kawai, S. Kojima, S. Watanabe and H. Suzuki, 1998.    Improved restriction landmark cDNA scanning and its application to global analysis of genes regulated by nerve grpth factor in PC12 cells.   Biochemica et Biophysica Acta 1399: 10-18. 1st dayRNA extraction using Dynabeads mRNA Direct kit (Dynal)

Grind 15 inmature barley grains (about 0.6 g) of each line or plant  in liquid nitrogen.

Usually mortor and pestle are suited. A mechanical cruasher, e.g. Multibeads Shocker MB301 (Yasui Kikai Corp.), is also applicable.Transfer the frozen powder to a 50-ml tube.  The powder can be stored at -80o C.  Morning, 2nd dayConditioning of Dynabeads Oligo(dT)25 　 1. Resuspend Dynabeads Oligo(dT)25 by gently flicking the tube or mixing on a roter. 2. Transfer 250 ul Dynabeads Oligo(dT)25 from stock tube suspension to an RNase-free 1.5 mL microcentrifuge tube placed in a magnet stand (MPC-M). 3. After 30 sec., remove supernatant.          Note: Do not leave Dynabeads Oligo(dT)25 unsuspended for a long time. 4. Remove tube from MPC-M and prewash Dynabeads Oligo(dT)25 by resuspending in 200 ul Lysis/Binding buffer.

Preparation of lysate  

5. Pour 5.0 ml Lysis/Binding buffer to the tube containing frozen powder made of barley grains. 6. Homogenize using Polytron or equivalent until complete lysis is obtained. 7.Centrifuge at 13,000 rpm for 2 min to remove debris. 8.Transfer supernatant to a clean new tube.          Note:The lysate can be frozen in liquid nitrogen and stored at -80o C for later use.

Direct mRNA isolation from crude lysate  

9. Place tube containing the Dynabeads Oligo(dT)25 (from step 4) in MPC-M and remove Lysis/Binding buffer. 10. Transfer tube from the MPC-M to another rack, and add lysate from step 8. 11. Mix Dynabeads Oligo(dT)25 with 1ml lysate and anneal by ratating on a roller or mixer for 3-5 min at room temperature. 12. Place tube in MPC-M for 2 min and remove supernatant. Transfer the tube to another rack. 13. Wash twice with 1 ml Washing buffer with LiDS at room temperature using MPC-M and once 1mL Washing buffer. Note:  Dynabeads Oligo(dT)25 must be mixed throughly in Washing buffer. Ensure the supernaqtant is completely removed between each washing step. 14. Add Elution solution (20 uL) and keep at 65o C for 2 min.   Immediately place the tube  in MPC-M and transfer supernatant containing mRNA to a new RNase-free tube. 15 Repeat 11 - 14 until there is no longer lysate.

Extracted mRNA concentration is 100 - 120 ug/mL.   Solution volume is 100 ul. A total of 10-12 ug mRNA may be obtained for each sample.

20 uL(2 ug mRNA) is used for RLCS.   Add Remaining solution (Remaining 80 uL mRNA solution should be EtOH precipitated and stored -20o C.)  

Afternoon, 2nd day cDNA synthesis using anchar primersPreparation of mixed anchor primers (a mixture of rlcsa, rlcsb and rlcsc)         rlcda: Biotin-gACTAgTTCTAgATCgCgAgCggCCgCCCTTTTTTTTTTTTTTTAA (46 mer)         rlcsg: Biotin-gACTAgTTCTAgATCgCgAgCggCCgCCCTTTTTTTTTTTTTTTAg (46 mer)         rlcdc: Biotin-gACTAgTTCTAgATCgCgAgCggCCgCCCTTTTTTTTTTTTTTTAC (46 mer)

Add dH2 O 33 uL to each anchor primer to make 100 pmol/uL (1.4 ug/uL) solution.

Heat 20 uL mRNA solution at 65o C for 10 min, then chill on ice.

Briefly spin a First-Strand Reaction Mix to collect solution at the bottom of the tube. Add 1 uL of DTT Solution (200 mM). Add 0.7 uL each anchor primers (0.7 ul x 3 = 2.1 uL).

Add heat-denatured RNA.  Pippet up and down to mix.

Incubate at 37o C for 1 hr.

Briefly spin a Second-Strand Reaction Mix. Transfer the first-strand reaction (33 uL) to this tube. Mix gently.

Incubate at 12o C for 30 min. and then at 22o C for 1 hr. incubate at 65o C for 10 min, and then store at 4o C.  

3rd dayAdd 100 uL PCI and votex briefly.Centrifuge at 13.000 rpm for 70 sec.Take upper phase and add 100 uL CI.Centrifuge at 13.000 rpm for 70 sec.Take upper phase and add 100 uL CI.

Ethanol precipitation using PeletPaint Co-presipitant Rinse twice by 70% Ethanol and once by 99.5% Ethanol and dry

Dissolve in 70 ul TE containing 20 ug/mL RNaseA and incubate at 37o C for 30 min.        (1 mL TE + 2 uL RNaseA stock of 10 mg/mL)

QIAquick purification

    Add 350 uL buffer PB to the DNA solution.     Place a QIAquick spin column in a provided 2 mL collection tube.     To bind DNA, apply the sample to QIAquick column and centrifuge (13,000 rpm) for 30-60 sec.     Dicard flow-through. Place the QIAquick column back in the same tube.     To wash, add 0.75 mL buffer PE to the column centrifuge (13,000 rpm) for 30-60 sec.     Dicard flow-through.     Place the QIAquick column back in the same tube.     Centrifuge (13,000 rpm)  the column for an additional 1 min.     Place a QIAquick spin column in a clean 1.5-mL collection tube.     To elute DNA, add 50 uL TE, let stand for 1 min., and then centrifuge (13,000 rpm).

estimate the cDNA concentration (usually about 1 ug)    

Digestion with Bam H I

     10X H Buffer     5 uL      Bam H I           2 uL         total        57 uL       incubate at 37o C for 3 hrs.

EtOH precipitation using PelletPaint Co-precipitant. rinse twice by 70% EtOH and once by 99.5% EtOH.

elute the precipitant in 5 uL TE.    

Preparation of gel holder (should be done during Digestion with Bam H I) Cut teflon-tubing (NICHIAS AWG-11, inner diameter: 2.4 mm) into 65 cm (cut a top at an angle of about 20o ) for gel holder.ｉｎｓｅｒｔ it into a glass tube from the bottom, and then a sharp tip of the tubing may exert form the top.Pull out the tip about 2 cm with a plier.Cut off the top of the tubing leaving about 1 mm in height.Push the top of the tubing onto a heated tip of a hand welder.After a few seconds, push the top of the tubing onto a flat and smooth surface to make a flange..Cut off the tubing at the bottom to adjust the length of the gel holder to 62 cm. The tubing is protruding 2 cm from the glass tube.　 4th day Labelling of cDNAthe DNA solution                   5 uL      For 2 samples    3 samples    4 samples[alpha-32P]dGTP (3,000 Ci/mmol)    1 uL          2.2          3.3          4.45X Buffer                          1.6 uL        3.52         5.28         7Sequenase ver. 2.0 (12 units/uL)   0.2 uL        0.44         0.66         0.9   total                           8 uL    Note: 5X reaction buffer of Sequenase ver. 2): 200 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 250 mM NaCl

Incubate at 27o C for 5 min. Add 42 uL TE.

QIAquick purification

    Add 250 uL buffer PB to the DNA solution.     Place a QIAquick spin column in a provided 2 mL collection tube.     To bind DNA, apply the sample to QIAquick column and centrifuge (13,000 rpm) for 30-60 sec.     Dicard flow-through. Place the QIAquick column back in the same tube.     To wash, add 0.75 mL buffer PE to the column centrifuge (13,000 rpm) for 30-60 sec.     Dicard flow-through. Place the QIAquick column back in the same tube.     Centrifuge (13,000 rpm)  the column for additional 1 min.     Place a QIAquick spin column in a clean 1.5 mL collection tube.     To elute DNA, add 50 uL TE, let stand for 1 min., and then centrifuge (13,000 rpm).  

Purification of target DNA using Dynabeads M-280 Streptavidin (Dynal)

Materials used:     Dynabeads M-280 Streptavidin (Dynal)  10 mg/mL     Magnet stand Dynal MPC-P-12     Above-mentioned DNA solution     Binding & Washing (B&W) solution (10 mM Tris-Cl, pH7.5, 1 mM EDTA, 2 M NaCl)

     For 1ml B&W      1 M Tris-HCl    10 uL      0.5 M EDTA       2 uL      5 M NaCl       400 uL      dH2O           588 uL  

Dynabeads M-280 Streptavidin should be washed before use to remove the 0.02% NaN3 added as a preservative. The washing procedure is facilitated by using a magnet (Dynal MPC).

1. Resuspend the Dynabeads M-280 Streptavidin by gently shaking the vial to obtain a homogeneous suspension. 2. Add 50 ul (0.5 mg) of Dynabeads M-280 Streptavidin (10 mg/mL) to a tube. 3. Place the tube in the Dynal MPC-P-12 for 2 min. Do not remove the tube from the magnet (Dynal MPC) during the separation process. 4. Remove the supernatant by aspiration with a pipette while the tube remains on the Dynal MPC. Avoid     touching the inside wall of the tube, where the Dynabeads attract to the magnet, with the pipette tip. 5. Remove the tube from the Dynal MPC. 6. Add the recommended buffer along the inside of the tube where the Dynabeads are collected. Use the same volume as in step 2 above and resuspend gently. 7. Repeat steps 3 to 5. 8. Resuspend the beads in 50 uL of B&W Buffer to a final concentration of 10 mg/mL (=10 ug/uL).

Streptavidin-biotin immobilization procedure

1. Add 50 ul washed Dynabeads M-280 Streptavidin to 50 uLl of the  DNA solution. 2. Incubate at 37o C for 1 hr occasional mixing by gently tapping the tubes. 3. Separate the Dynabeads M-280 Streptavidin, now coated with the biotinylated DNA fragments, using a Dynal MPC. Leave the tube/tray in the Dynal MPC for 2 minutes. 4. Wash twice with 50 uL B&W Buffer and twice with 50 uL 1X H buffer containing 0.1% BSA and 0.1% Triton-X , using a Dynal MPC as described above. 5. Digest with Not I

                    10x H buffer            5 uL                     Not I (10 u/ul)         5                     0.1% BSA                5                     0.1% Triton-X           5                     dH2 O                   25                         total              45

                                 37 C for 2 hrs.  

6. Set in a Dynal MPC. Leave the tube/tray in the Dynal MPC for 2 minutes.   Collect supernatant (50 uL).EtOH precipitation using PelletPaint Co-precipitant.rinse twice by 70% EtOH and once by 99.5% EtOH.Dry.elute in 6 uL TE

Add 1 uL 6X loading buffer.  

Preparation of 1-D Gel (should be done during Not I digestion) Connect a 5 ml plastic syringe fitted with a three-way stopcock and the gel holder with 3 cm of silicon tubing.Suck up the gel solution gradually to reach the height of 61 cm, and tap on the glass by a finger.Close the stopcock and set it on a double buret clamp on a support stand.Wait 10 min. to solidify the gel.Turn the stopcock to make air free.Remove the stopcock and silicon tubing.If the gel does not reach the height of 61 cm, put on the gel solution up to the height of 61 cm using a 1 ml syringe with a L-needle (19gauge, right angle cut, 5 cm long) and wait 5 min.Put 1-D buffer on the gel to avoid drying.Add 350 ml of 1x 1-D buffer to anodal tank (bottom) and fix the gels on the apparatus (Bio-craft) .　 1-D ElectrophoresisFill the cathodal tank (top) with 300 ml of 1x 1-D buffer.Remove bubbles on the top of each gel.Conduct electrophoresis at 115 V (initially 5 mA) for 40 hrs. (until the center of BPB reaches 38 cm and that of XC reaches 18 cm fromthe top of the gel).　 5th day Preparation of 2-D GelSiliconize one side (the side with a tapered edge) of a glass plate.Set up the gel casting apparatus (Bio-craft).Mix a gel solution and degas it by vacuum with a sonicator for 5 min.Cover the top of the gel with Kim Towel wetted by 1 x TBE containing 1% glycerin to keep wet.　 Morning, 6th day in situ digestion After stopping the 1-D electrophoresis remove the cathodal top buffer with aspirator and take out each gel holder.Expel the gel slowly using a shortened yellow tip on a 1 ml syringe filled with 0.05% BPB water 10x high buffer (HB)Cut off the bottom 12 cm of the gel at 45o .Expel the gel slowly using a shortened yellow tip on a 5 ml syringe filled with 0.05% BPB water 10x high buffer (HB)Cut off the top 15 cm of the gel at 90o .Soak the gel in 50 ml tube containing 45 ml of 1x HB.Shake the tube gently and equilibrate the gel for 10 min.Change the buffer and equilibrate the gel once again for 10 min.Pour the equilibrated gel into a stainless tray.Place the gel noodle (35 cm long corresponding with 180 bp to 6 kb) in a plastic tray of 11(W) x 350(L) x 3(D) mm.Remove any trace of the buffer.Pour 3 ml of 1x HB containing 1000 units of Hinf I and 0.01% BSA (enzyme solution).Seal the tray with Saran Wrap (incubate at 37o C for 2 hrs.Meanwhile, set up the 2-D electrophoresis apparatus (Bio-craft).Fill the electrode tanks with 1x TBE buffer (3 L for the bottom tank).  Afternoon, 6th day Running 2-D gel electrophoresisRinse the top of the 2-D gel with 1x TBE and wipe with Kimwipe after in-situ digestion with Hinf IExpel the gel noodle from into a 50 mL tube containing 45 mL of 1x TBE.Equilibrate for 10 min.Discard the buffer and pour the gel noodle onto a plastic board.Transfer the gel onto the top of 2-D gel.Connect agarose noodle gel and 2-D gel with 3 mL of connection agarose gel solution using a 5 ml syringe with 20 G needle.Overlay dye agarose gel solution using a 5 mL syringe with 20 G needle.Fill the electrorode tank with 1x TBE buffer (1.5 L for the upper tank).Run electrophoresis at 100 Volts for 40 hrs until the BPB and XC reaches about 38 and 20 cm from the top, respectively.   (BPB indicates about 60 bp and XC indicates about 330 bp)  Morning, 8th day Gel dryingStop the 2-D electrphoresis, and dismantle the apparatus.Cover the gel with a piece of filter paper (3 MM, 345 mm x 425 mm).Cut off the gel outside of the filter paper.Take up the gel with the filter paper, and lay it down on two pieces of filter paper (3 MM, 360 mm x 440 mm) with the gel side up.Overlay Saran Wrap (45 cm wide) onto the gel, and then a piece of filter paper (3 MM, 360 mm x 440 mm) on them.Cut off an excessive area of the Saran Wrap remaining 5 mm edge.Set the gel on a gel dryer (Bio-Rad, Model 583).Dry up the gel at 60o C for 1.2 hr.　 AutoradiographySet an intensifying screen (on the bottom), the dried gel, and then a piece of filter paper (3M) on them in a film cassette.ｉｎｓｅｒｔ an X-ray film (KODAK X-OMAT AR) between the gel and the intensifying screen in a dark room.Perform autoradiography at -80o C for 60 hr or longer.Warm the cassette up to the ambient temperature.Develop, fix and dry the X-ray film in a dark room.   Now you can examine the RLCS profile.