Analysis of RNA by Primer Extension

INTRODUCTION

Primer extension is used chiefly to map the 5′ termini of mRNAs. A preparation of polyadenylated mRNA is first hybridized with an excess of a single-stranded oligodeoxynucleotide primer, which is complementary to the target RNA and radiolabeled at its 5′ terminus. Reverse transcriptase is then used to extend the 3′ end of the primer. The size of the resulting cDNA, measured by denaturing polyacrylamide gel electrophoresis, is equal to the distance between the 5′ end of the priming oligonucleotide and the 5′ terminus of the target mRNA.

MATERIALS

Ammonium acetate (10 M)

Carrier RNA (yeast tRNA)

Chloroform

DNA markers, radiolabeled, for gel electrophoresis

DTT (dithiothreitol) (1 M)

Ethanol

Formamide loading buffer

Input RNA to be analyzed

KCl (1.25 M)

Oligonucleotide primer

Phenol

Polynucleotide kinase

Primer extension mix

Protein inhibitor of RNase

Reverse transcriptase

Sodium acetate (3 M, pH 5.2)

TCA (trichloroacetic acid) (1% and 10%)

TE (pH 7.6)

[γ-³²P]ATP (10 mCi/ml, 7000 Ci/mmole)

METHOD

• 1. Phosphorylate the oligonucleotide primer in a reaction containing:Incubate the reaction for 60 minutes at 37°C.

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  The final concentration of radiolabeled ATP in the reaction should be approx. 30 nM.

• 2. Stop the kinase reaction with the addition of 500 μl of TE (pH 7.6). Add 25 μg of carrier RNA.

• 3. Add 400 μl of equilibrated phenol (pH 8.0) and 400 μl of chloroform (or 800 μl of commercial phenol:chloroform [1:1]). Vortex vigorously for 20 seconds. Separate the aqueous and organic phases by centrifugation for 2 minutes in a microcentrifuge.

• 4. Transfer the aqueous layer to a fresh sterile microcentrifuge tube and extract with 800 μl of chloroform. Vortex vigorously for 20 seconds. Separate the aqueous and organic phases by centrifugation for 2 minutes in a microcentrifuge. Again transfer the aqueous layer to a fresh sterile microcentrifuge tube.

• 5. Repeat Step 4.

• 6. Add 55 μl of sterile 3 M sodium acetate (pH 5.2) and 1 ml of ethanol to the aqueous layer from Step 5. Mix by vortexing and store the solution for at least 1 hour at -70°C.

• 7. Collect the precipitated oligonucleotide primer by centrifugation at maximum speed for 15 minutes at 4°C in a microcentrifuge. Remove and discard the radioactive supernatant. Wash the pellet in 70% ethanol and centrifuge again. Discard the supernatant and dry the precipitate in the air. Dissolve the precipitate in 500 μl of TE (pH 7.6).

• 8. Count 2 μl of radiolabeled oligonucleotide primer in 10 ml of scintillation fluid in a liquid scintillation counter. Calculate the specific activity of the radiolabeled primer assuming 80% recovery. The specific activity should be approx. 2 x 10⁶ cpm/pmole of primer.

• 9. Mix 10⁴ to 10⁵ cpm (20-40 fmoles) of the DNA primer with 0.5-150 μg of the RNA to be analyzed. Add 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol. Store the solution for 60 minutes at -70°C, and then recover the RNA by centrifugation at maximum speed for 10 minutes at 4°C in a microcentrifuge. Wash the pellet with 70% ethanol and centrifuge again. Carefully remove all of the ethanol, and store the pellet at room temperature until the last visible traces of ethanol have evaporated.

• 10. Resuspend the pellets in 8 μl of TE (pH 7.6) per tube. Pipette the samples up and down several times to dissolve pellets.

• 11. Add 2.2 μl of 1.25 M KCl. Vortex the samples gently and then deposit the fluid in the base of the tubes by centrifuging for 2 seconds in a microcentrifuge.

• 12. Place the oligonucleotide/RNA mixtures in a water bath set at the appropriate annealing temperature. Incubate the samples for 15 minutes at the optimum temperature, as determined in preliminary experiments.

• 13. While the oligonucleotide and RNA are annealing, supplement an aliquot of primer extension mix with dithiothreitol and reverse transcriptase as follows: Thaw a 300-μl aliquot of primer extension mix on ice and then add 3 μl of 1 M dithiothreitol and reverse transcriptase to a concentration of 1-2 units/μl. Add 0.1 unit/μl of protein inhibitor of RNase, gently mix by inverting the tube several times, and store it on ice.

• 14. Remove the tubes containing the oligonucleotide primer and RNA from the water bath and deposit the fluid in the base of the tubes by centrifuging for 2 seconds in a microcentrifuge.

• 15. Add 24 μl of supplemented primer extension mix to each tube. Gently mix the solution in the tubes and again deposit the liquid at the tube bottoms by centrifugation.

• 16. Incubate the tubes for 1 hour at 42°C to allow the primer extension reaction to proceed.

• 17. Terminate the primer extension reactions by the addition of 200 μl of TE (pH 7.6), 100 μl of equilibrated phenol (pH 8.0), and 100 μl of chloroform. Vortex for 20 seconds. Separate aqueous and organic phases by centrifugation for 4 minutes at room temperature in a microcentrifuge.

• 18. Precipitate the nucleic acids by the addition of 50 μl of 10 M ammonium acetate and 700 μl of ethanol. Mix well by vortexing and incubate ethanol precipitations for at least 1 hour at -70°C.

• 19. Collect the precipitated nucleic acids by centrifugation for 10 minutes at 4°C in a microcentrifuge. Carefully rinse the pellets with 400 μl of 70% ethanol. Centrifuge again for 5 minutes at 4°C and remove the 70% ethanol rinse with a pipette. Store the open tubes at room temperature until all visible traces of ethanol have evaporated.

• 20. Dissolve the nucleic acid precipitates in 10 μl of formamide loading buffer. Pipette the samples up and down to assist resuspension.

• 21. Heat the samples for 8 minutes at 95′C. Then plunge the tubes into an ice-water bath and immediately analyze the primer extension products by electrophoresis through a denaturing polyacrylamide gel.

• 22. After the tracking dyes have migrated an appropriate distance through the gel, turn off the power supply and dismantle the electrophoresis setup. Gently pry up one edge of the larger glass plate and slowly remove the plate from the gel. Cut off one corner of the gel for orientation purposes.

• 23. If a polyacrylamide gel 1.0 mm in thickness was used, fix the gel in TCA. Transfer the glass plate containing the gel to a tray containing an excess of 10% TCA. Gently rock or rotate the tray for 10 minutes at room temperature.

  This step is not necessary if a thin gel (0.4-mm thickness) was used. In this case, proceed to Step 26.

• 24. Pour off the 10% TCA solution and replace it with an excess of 1% TCA. Gently rock or rotate the tray for 5 minutes at room temperature.

• 25. Pour off the 1% TCA solution and briefly rinse the fixed gel with distilled deionized H₂O. Lift the glass plate together with the gel out of the tray and place them on a flat bench top. Apply paper towels or Kimwipes to the sides of the gel to remove excess H₂O.

• 26. Cut a piece of Whatman 3MM filter paper (or equivalent) that is 1 cm larger than the gel on all sides. Transfer the gel to the filter paper by laying the paper on top of the gel and inverting the glass plate.

• 27. Remove the plate and dry the gel on a heat-assisted vacuum-driven gel dryer for 1.0-1.5 hours at 60°C.

• 28. Establish an image of the gel using autoradiography or phosphorimaging.