Primer Extension analysis of total yeast RNA (From Steve Hahn Last modified 9/19/06 by SKM)

First kinase the oligos using following protocol:.

Labeling oligonucleotides with 32P ATP

Wear gloves throughout and work in radiation area. Monitor area before and after use.

Mix the following in an eppendorf tube:

1. 0.5 microgram oligonucleotide dissolved in H2O.
2. 3 microliters 10x kinase buffer.
3. 2 microliters 32P ATP from ICN (>5000 ci/mmole).
4. H20 so that the final volume is 30 microliters.

Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 deg.
Purify labeled Oligonucleotide away from unincorporated ATP
Currently, we use mini Quick Spin Oligo Columns (#1 814 397) from Roche to purify the labeled oligonucleotide.
Prepare the column according to the manufactuer's instructions by centrifugation of the resuspended matrix for 1 min @ 1000 x g.
Insert column into a new eppendorf tube and add oligo labeling reaction, adding slowly to center of column. Centrifuge 1000 x g for 4 min.
Recover purified labeled oligo. For most applications, add 70 microliters TE to the 30 microliters recovered for a total of 100 microliters.
Quantitate radioactive incorporation by counting 1 microliter of a 1/10 diluted sample. Expect between 20 -100 million cpm total.

10x Kinase Buffer

0.5 M Tris pH 7.6
0.1 M MgCl2
50 mM DTT

Primer Extension analysis of total yeast RNA :
1. Mix the following in a 1.5 ml eppendorf tube:
a. 20-80 micrograms yeast RNA dissolved in H20 (the amount will depend on the level of expression for the gene of interest)
b. 2.0 ml 5X annealing buffer c. 200,000 - 400,000 CPM Kinased primer (typically about 1 ng) d. 1ml of RNAse Inhibitor d. H20 to a final volume of 10 microliters.

2. Incubate tubes for 1 minute in a boiling water bath and then immediately transfer to 37oC bath. Incubate for 45-60 minutes to allow annealing of primer and RNA.

3. Spin tubes briefly in a microfuge to bring down any condensation on the sides and top of tubes.

4. Add 20 ml of AMV Reverse Transcriptase (Roche only) Synthesis Mix. Incubate 30 min at 37 oC.

5. To terminate the reaction, add 1 ml of 0.5 M EDTA, pH 8.0 and Add 5 ml of RNAse A (10mg/ml) and incubate at 37 oC for 10 min.

6. Finally, add 150 ml of TE, and extract with 200 ml phenol/Chloroform.

7. Ethanol precipitate samples by adding 3 volumes ethanol, 1/10th vol NaOAc and 1 ml Glycogen (20mg/ml). Spin, and wash pellets with 80% ethanol. Air Dry pellets for 15-20 min.

8. Add 10 ml Formamide sequencing gel loading buffer and mix by vortexing.

9. Heat samples 90oC for 2 min and then chill rapidly on ice. Load samples to Urea/acrylamide sequencing gel.

10. Before loading the gel, DON’T FORGET to PRERUN the gel at 1300V for 15-30 min.
Reverse Transcriptase Synthesis Mix:
4 microliters 5X RT buffer from Roche. 0.3 microliters each dNTP (10 mM dATP, dCTP, dGTP, TTP) 13.3 microliters H2O 1.5 microliters M-MLV Reverse Transcriptase (25 units/microliter Roche)

5X Annealing Buffer: 1 ml
25 mM Tris pH 8.3 25 ml 1 M Tris 8.3 375 mM KCl 0.375 ml 1 M KCl 5 mM EDTA 20 ml 0.25 M EDTA and make up to 1 ml.

MAPPING TRANSCRIPTION INITIATION SITES WITH PRIMER EXTENSION

The primer extension reaction is used to determine the start site(s) of RNA transcription for a particular gene. One uses a radiolabelled primer that is complementary to a region towards the 5' end of the transcript, and reverse transcribes a single stranded DNA molecule towards the 5' end of the RNA. The size of the labelled single-stranded DNA is then determined on a sequencing gel relative to an M13 ladder.

We typically use at least two sources of total cellular RNA: HeLa cells, and either K562 or HEL cells. RNA isolated from tissue can also be used.

We also perform the reaction with two different primers, one just inside the coding region, one upstream of exon 1 if that sequence is known.

Procedure

1. Radiolabel each primer with T4 polynucleotide kinase.
2. Mix about 5ng (about 100 cpm) of primer to each RNA in a small volume (10 – 30 ml) of annealing buffer. You may need to co-precipitate RNA with the primer. If so, avoid vacuum desiccation since the RNA may not go back into solution. The amount of RNA to use varies with its copy number. Try a range, such as 1, 10, and 100 mg of total RNA.
3. Annealing: Heat to 85 oC for 10 minutes and transfer to a 30 oC water bath overnight. Precipitate in a dry ice/ethanol bath by adding 170 ml H2O and 400 ml absolute ethanol. Wash the pellet once with 70-80% ethanol. Evaporate with open cap on the lab bench or fume hood to remove last trace of ethanol.
4. Reverse Transcription: Resuspend the annealed nucleic acids in 20 ml of reverse transcription buffer. Add 40 U of AMV reverse transcriptase for 90 minutes, at 30 or 42oC. To terminate the reaction, add 1 ml of 0.5 M EDTA, pH 8.0 & 1 ml of DNAse free RNAse for 30 minutes, 37oC. Finally, add 150 ml of TE with 100 mM NaCl in it, and extract with 200 ml phenol/CHCl3. Ethanol precipitate the cDNA and wash the pellet once with 70-80% ethanol. Evaporate the remaining ethanol with the microfuge cap open on the lab bench.
5. Electrophoresis: Dissolve the cDNA in 4 ml TE. Add 6 ml of sequencing stop dye (formamide dye mix). Heat the samples for 5 minutes at 95°C before loading on sequencing gel. Load M13 control sequencing reactions in adjacent lanes as molecular weight markers.

RECIPES

Annealing buffer 40 mM PIPES (pH 6.4) 1 mM EDTA 0.4 M NaCl 80% formamide

Reverse transcription buffer 50 mM Tris-HCl, pH 7.6 35 (or 60 mM) KCl 10 mM MgCl2 1 mM each dNTP 1 mM DTT 1 U RNasin