8 replies to this topic

[marek82313]

Hello everybody,

I've just performed my first 5' RACE and I'm really puzzled by the results.
Here is the protocol I've used:
1 cytoplasmic RNA was purified with trizol
2 First Strand cDNA was prepared using random primers
3 cDNA was purified by ammonium acetate/EtOH precipitation
4 A polyC tail was added to the cDNA 3' End
5 The 5' Race was performed using a combination of PolyG-tailed primers, adaptor primers and gene specific primers (forward or reverse, see the figure below, 1-F and 2-F reaction were performed using forward primers and 1-R and 2-R were performed using reverse primers)
6 1% of the product was loaded in a 1.5% gel

Both the reaction setted up with forward or reverse primers amplified for a band 300-400 bp long, only the reaction setted up with reverse primer amplified for a band about 125 bp long. Faint bands can be seen in both the reaction products.
The annealing of the primers was performed at 60°C (3 cycles) and the at 65°C (32 cycles). These temperatures were tested previously performing a PCR on a fragment of known length that was polyC tailed previously. The reaction produced only a specific fragment.

What do you think?  

[pcrman]

I am confused. For 5' race, what adpator primer has been added to the 5'end of the cDNA, what are 1F, 1R, 2F and 2R reactions? What do you expect to see and what is wrong with the products?

[marek82313]

Ok, sorry, I hadn't explained well my protocol. The protocol is a modification of the one explained in Maniatis.
I don't know neither the orientation nor the length of the transcripts I'm looking for, I know only the locus from these RNA are transcribed, so I started reverse transcribing the RNA using random primers (since I have to look for various loci I preferred reverse transcribe most of the RNA and amplify later from the first strand cDNA using specific primers). I expect that at least one molecule of target RNA was reverse transcribed (and so it was, in fact I checked it using specific primers to amplify the cDNA and I found a band of the expected length).
Now I need the full length cDNA, so, I started from the first strand cDNA obtained using random primers, and I added a polyC tail to the 3' end (that's complementary the 5' end of the template sequence, that's the RNA) and I performed a PCR using a primer containing a polyG sequence bound to an oligonucleotide of known sequence (that should anneal to the polyC tail of the cDNA, let's call this primer polyG-forward), an adaptor primer which sequence correspond to the one of the oligonucleotide bound to the polyG tail (let's call this
Adaptor), and a specific primer (designed on DNA strand, in this case I have 2 primers, forward or reverse since I don't know from which strand the RNA is transcribe, let's call these primers 1-F and 1-R and 2-F and 2-R).

The reactions are setted up so:

Reaction Mix 1-F
cDNA
polyG-forward
Adaptor
Primer 1-F

Reaction Mix 1-R
cDNA
polyG-forward
Adaptor
Primer 1-R

Reaction Mix 2-F
cDNA
polyG-forward
Adaptor
Primer 2-F

Reaction Mix 2-R
cDNA
polyG-forward
Adaptor
Primer 2-R

you can see a picture depicting the reaction here:

http://www.dbsm.unin...ges/Molecu3.JPG

I'm puzzled because I thought I would have find a longer product, and I didn't expect to see some supplementary bands (aspecifics?) between my products.
I just want know if someone experienced something similar and found out that is due to a wrong reaction set up.
Maybe what I obtained is correct, I really don't know because we never performed 5' race before and we have no preliminary data about the possible length of the transcript.
I know is all twisted but...

[pcrman]

Now i got you. The adaptor is added to the 3 end of the cDNA (5' of the mRNA). Your problem is that you obtained multiple bands while you only expected one. This does not necessarily mean that the results are not right. For any gene, transcription can start from many different locations, with one or more than one being the dominant. So when you do 5' RACE, you may get multiple bands. the same is true if you do a primer extension. To verify whether those bands really represent 5' end of different length, you can sequence the bands.

BTW, which genome you are working with?

[marek82313]

Thank you for your help pcrman,

I started a series of experiment to define if the 300 bp band is due to aspecific annealing or what using different combinations of primers (polyG tailed, adaptor and and gene specific primers that should anneal with the p53 mRNA). I should have performed them before, sigh  


I'm working with the old, dear, beloved human genome. In particular these are cytoplasmic RNAs from the 293F and HT-1080 cell lines.

[mastermi]

Whenever you get distinct bands after 5'Race, I would simply cut out all of them and send them for sequencing (or subclone them in pUC for sequencing).
When you got the sequencing results you can easily see if the amplified fragment has got anything to do with the RNA of your interest. That is for sure the fastest way to check it...

[pcrman]

What is your purpose for doing 5 RACE. If you work with human genome, almost all genes and their 5' end are known. Of course there are non-standard transcription start site for many genes. There is a database called DBTSSwhich contains all possible TSSs determined by 5 RACE for all genes. At least you can have a look at the database to see whether you should get multiple 5' RACE products or not.

[marek82313]

For mastermi

sequencing is for sure the only way to check the products I've obtained are the specific ones, we were puzzled because the RACE performed with 3 distinct loci produced a band of, apparently, the same length, so we are trying to find out what's appening, the first thing we thought is that we have a problem with the reaction set up.


For pcrman
You are right pcrman, but, to date, there's no proof that the loci that I'm studying are transcribed, only few EST in GenBank can be found.  I used various promoter prediction softwares to determine the possible localization of promoters or transcription start sites that can partecipate to the transcription of these sequences, but all the softwares I used produced different results    ...

Does anybody know reliable promoter-prediction software, transcription start site prediction softwares or CpG island prediction software?

[pcrman]

so you are working to identify a potential new gene which is very interesting. Yes you can analyze the 5'flanking sequence to see if there is a promoter. For promoter prediction I like BDGP: Neural Network Promoter Prediction very much. It does a much better job than other programs. There are many CpG island prediction programs on the net. UCSC genome browser also has a CpG track. If there is a CpG island at the 5' end of your gene, it is most likely a promoter. If there is not, then promoter prediction program will have a hard time guessing where a promoter is.