The Primer Extension Assay

1. Mix 5 µL 10× T4 PNK buffer with 2.5 picomoles of purified oligonucleotide and 5 µL (∼10 pmoles) of [γ-³²P]ATP. Add dH₂O to a final volume of 49 µL.

2. Add 1 µL (10 Units) of T4 polynucleotide kinase.

3. Incubate for 45–60 min at 37°C.

4. Heat-inactivate the kinase for 10 min at 68°C.

Unincorporated [γ-³²P]ATP molecules should be removed from the primer preparation to prevent them from interfering with the primer extension reactions and, in particular, with gel electrophoresis and autoradiography. Unincorporated nucleotides can be removed by differential ethanol precipitation in the presence of ammonium acetate (see Purification of Radiolabeled Oligonucleotides by Precipitation with Ethanol [Sambrook and Russell 2006]) or by column chromatography (as described here).

• 5. Add 1 µL of 5 m NaCl to the heat-inactivated reaction sample.

• 6. Prewash a Stratagene NucTrap column by applying 70 µL of STE buffer.

• 7. Extend the plunger of a 10-cm³ BD syringe with a Luer-Lok tip and screw the syringe onto the column to form a seal between the column and the syringe according to the manufacturer’s protocol. Do not screw the syringe on too tightly.

• 8. Force the buffer down the length of the column until a small drop exits at the end. Use the prewetted column within 5–10 min.

• 9. Attach the column to the Beta Shield according to the manufacturer’s instructions.

• 10. Apply the radioactive solution to the column, attach the extended syringe, and slowly (25–35 sec) push the sample through the column with the plunger. Collect the flowthrough in a microfuge tube. The unincorporated nucleotides will remain in the resin and the radiolabeled primer will pass through the column into the tube.

• 11. Apply 70 µL of STE solution to the column, push through with the syringe, and collect the flowthrough in the same tube.

• 12. Dilute the primer solution with an additional 140 µL of STE buffer.

• 13. Quantitate the radioactivity by scintillation counting (Cerenkov counting; i.e., in the absence of scintillation fluid). Cerenkov measurements vary from machine to machine, but roughly 50,000–100,000 cpm per μL should be obtained in the final primer solution.

  • See Troubleshooting.

• 14. Store the primer at –20°C. It can usually be used for up to 1 mo.

Nuclease-free buffers and reagents and RNase inhibitors can be used for primer extension reactions, but they generally are not necessary. Extensive degradation would be needed to affect the results of a primer extension assay noticeably, because only the 5′ end of each mRNA molecule needs to be intact for the primer extension assay to succeed.

• 15. Prepare 5× PE buffer and RT buffer (store at –20°C).

Negative control RNAs should be included whenever possible and are essential for mapping unknown start sites. A tRNA or yeast RNA control yields less information but can be used as an additional control. Ideally, negative control RNA should be prepared from a cell line or tissue that does not express the RNA being measured and, ideally, this cell line should be from the same species as the experimental RNA. If possible, RNAs prepared from multiple positive and negative cell lines or tissues should be tested. Without appropriate negative controls, mapping of a novel start site will be less compelling.

• 16. Add 1 µL of radiolabeled primer (∼50,000–100,000 cpm as measured by Cerenkov counting) to 10–60 µg of total RNA (or ∼2–5 µg of poly(A)⁺ mRNA). The optimal amount of RNA to use for a primer extension reaction will vary and may need to be determined empirically. In general, the success of the procedure is less dependent on the amount of RNA than on other variables, such as the specific activity of the primer, the annealing temperature, and the presence or absence of RNA secondary structures. During initial experiments, 30 µg of total cellular RNA or total cytoplasmic RNA is an appropriate amount to use.

• 17. Add 3 m sodium acetate (pH 5.2) to a concentration of 0.3 m, followed by 2.5 volumes of ethanol. Incubate in dry ice for 10 min or at –20°C for 30 min to precipitate the RNA and primer.

• 18. Pellet the RNA and primer by centrifugation at 14,000g for 10 min in a microfuge.

• 19. Carefully remove the supernatant and dry the pellet in a SpeedVac or by evaporation on the bench top. Virtually all of the radioactivity should be present in the pellet, with trace amounts in the supernatant.

  • See Troubleshooting.

• 20. Suspend the dried pellet in 8 µL of TE buffer. The pellet will not suspend in this small volume of buffer by vortexing because the buffer will not remain at the bottom of the tube. The preferred method for pellet suspension is to “flick” the bottom of the tube repeatedly with one’s finger. Alternatively, the pellet can be dissolved by repetitive pipetting of the solution with a pipetman.

• 21. Add 2 µL of 5× PE buffer to the tube. Mix and centrifuge briefly.

  • The 5× PE buffer should not be added until the RNA has dissolved in the TE because the RNA will dissolve much more slowly in the high-salt PE buffer.

• 22. Anneal the primer to the RNA by incubating 90 min at the empirically determined temperature. During the incubation, centrifuge the samples briefly every 20 min, so that the solution does not condense at the top of the tube.

  • A good starting point is to test temperatures of 37, 45, 60, and 68°C. Some primers work well if the hybridization reactions are first heated to 68°C, then allowed to cool slowly to room temperature in a metal block.

• 23. Prepare a solution containing 39.5 µL of RT buffer and 0.5 µL of MMLV reverse transcriptase per each reaction. Optionally, add 50 µg/mL actinomycin D to the RT buffer. Since Pipetmen are not perfectly accurate, it is best to make up a larger amount of the solution than is needed.

  • Actinomycin D inhibits the synthesis of double-stranded DNA by reverse transcriptases and is sometimes included in the reverse transcriptase reactions to prevent the synthesis of hairpin molecules. Hairpin molecules are rarely a problem with short synthetic oligonucleotide primers, unless the expected extension products are also very short.

• 24. Add 40 µL of RT mix to each reaction tube. Mix, but do not vortex. Centrifuge briefly.

• 25. Incubate for 60 min at 37°C. Higher temperatures (e.g., 42, 45, 55°C) can be used in an attempt to diminish unwanted effects of the RNA secondary structure.

• 26. Stop the reaction and precipitate the nucleic acids by adding 6 µL of 3 m sodium acetate and 150 µL of ethanol. Vortex well. Chill on dry ice for 10 min or at –20°C for 30 min.

• 27. Centrifuge for 10 min. A small white pellet should be visible on the bottom of the tube. Remove the ethanol. Blot the top of the tube on a Kimwipe to remove excess liquid. Most of the radioactivity should remain in the pellet.

• 28. Dry the pellet well in a SpeedVac evaporator.

29. Suspend the pellet in 4 µL of formamide loading dye and 2 µL of 0.1 m NaOH.

• At this point, the amount of radioactivity in each tube should still be similar to the amount added to the reactions in Step 16: If the amount is less, the precipitations probably did not work well or the pellets were lost.

30. Boil for 2 min to denature the DNA/RNA hybrids. Centrifuge briefly.

31. Load 3 µL of each sample onto an 8% denaturing polyacrylamide sequencing gel, after rinsing the wells of the gel.

32. Run the gel until the bromophenol blue dye is near the bottom. (The goal is to have the excess labeled primer migrate near the bottom of the gel.)

33. Dry the gel and expose it to XAR-5 film in the presence of an intensifying screen, or analyze on a phosphorimager. Strong signals on a film should be detectable with an overnight exposure.

• See Troubleshooting.