5' RACE products appearance on 1.5% agarose gel
Posted 09 April 2009 - 09:58 AM
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?
Posted 09 April 2009 - 08:07 PM
Posted 10 April 2009 - 02:14 AM
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
Reaction Mix 1-R
Reaction Mix 2-F
Reaction Mix 2-R
you can see a picture depicting the reaction here:
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...
Posted 10 April 2009 - 08:37 PM
BTW, which genome you are working with?
Posted 11 April 2009 - 02:04 AM
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.
Posted 11 April 2009 - 03:53 AM
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...
Posted 11 April 2009 - 06:34 AM
Posted 11 April 2009 - 08:34 AM
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.
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?
Posted 11 April 2009 - 08:55 AM