MAPPING TRANSCRIPTION INITIATION SITES WITH PRIMER EXTENSION

The primer extension re action 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

Radiolabel each primer with T4 polynucleotide kinase. Mix about 5ng (about 100 cpm) of primer to each RNA in a small volume (10 � 30 m l) of annealing buffer. You may need to co-prec ipitate 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 m g of total RNA. Annealing: Heat to 85 ° C for 10 minutes and transfer to a 30 ° C water bath overnight. Precipitate in a dry ice/ethanol bath by adding 170 m l H2O and 400 m l 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. Reverse Transcription: Resuspend the annealed nucleic acids in 20 m l of reverse transcription buffer. Add 40 U of AMV reverse transcriptase for 90 minutes, at 30 or 42 ° C. To terminate the reaction, add 1 m l of 0.5 M EDTA, pH 8.0 & 1 m l of DNAse free RNAse for 30 minutes, 37< /FONT>° C. Finally, add 150 m l of TE with 100 mM NaCl in it, and extract with 200 m l 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. Electrophoresis: D issolve the cDNA in 4 m l TE. Add 6 m l 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 50 m g/ml actinomycin D