Failed RT-PCR amplification of 3-&5''-ligated small RNAs - (Jun/10/2008 )
Hi everyone,
I am working on cloning small RNAs using the Bartel Lab protocol. I have done the 3' and 5'-ligation and reverse-transcriped the small RNAs.
However, i keep failed to PCR-amplify the ligated small RNAs, which are ~52-60bp. I keep having an unknown pcr products, which are about 30-40bp bright smear on 12% polyacrylamide gel stained with SYBR gold. Only very faint smear of ~52-60bp is seen. I think because of this strong unknown background have make the PCR failed.
I try add in 5% DMSO, but the result is still the same.
Any suggestion to solve this problem??
Hi,
some time ago I made a library with the protocol from the Bartel lab, but I was lucky and didn't have problems. The pcr-amplified library looked like a faint, slightly smeared, single band. I don't know where your artefacts come from, perhaps something wrong with the selection of the fractions after the ligations steps? You surely have seen the gallery of possible artefacts that come with with the protocol, when you download it.
There was a previous post on this matter. Have a look, maybe you can contact the person that started the topic and he will be a greater help.
http://www.protocol-online.org/forums/inde...ost&p=97718
Regards, good luck
some time ago I made a library with the protocol from the Bartel lab, but I was lucky and didn't have problems. The pcr-amplified library looked like a faint, slightly smeared, single band. I don't know where your artefacts come from, perhaps something wrong with the selection of the fractions after the ligations steps? You surely have seen the gallery of possible artefacts that come with with the protocol, when you download it.
There was a previous post on this matter. Have a look, maybe you can contact the person that started the topic and he will be a greater help.
http://www.protocol-online.org/forums/inde...ost&p=97718
Regards, good luck
Hi Andrea,
Sorry for late reply. Yew, i have review all my gel photos and compared to the Bartel online protocol, i think i should have cut out the correct size of the ligation products.
I have done a PCR that have no ligated RNA added in. I suspect the primers problem. I put the 3'primer and 5'primer in separate tubes to check whether the primer itself form dimers during PCR. The result showed that the primer itself do not form dimer. Then i did the same but with both primers in a tube, and the 30-40bp possible primers amplified products was observed.
After that, i increased the annealing temperature from 50C to 62C. No possible primer dimers was observed in PCR with 62C annealing temperature. But at that high temperature, the RT-PCR surely didnt work as i have repeated several times.
I rechecked again n again on the Lau journal and Bartel and Ambros labs protocols. The primers i synthesized are exactly the same as the protocols above mentioned. Eventually, i come to this conclusion that, the quality of the primers is probable not good which result in this unknown pcr products. What is your opinion? I try synthesize new sets of primers from other company.
While waiting, i cut out the expected RT-PCR products (50-bp) from the gel and do re-amplification. Now i have done the concatamerization and trying to clone it. Hopefully everything will be fine.
some time ago I made a library with the protocol from the Bartel lab, but I was lucky and didn't have problems. The pcr-amplified library looked like a faint, slightly smeared, single band. I don't know where your artefacts come from, perhaps something wrong with the selection of the fractions after the ligations steps? You surely have seen the gallery of possible artefacts that come with with the protocol, when you download it.
There was a previous post on this matter. Have a look, maybe you can contact the person that started the topic and he will be a greater help.
http://www.protocol-online.org/forums/inde...ost&p=97718
Regards, good luck
Hi Andrea,
Sorry for late reply. Yew, i have review all my gel photos and compared to the Bartel online protocol, i think i should have cut out the correct size of the ligation products.
I have done a PCR that have no ligated RNA added in. I suspect the primers problem. I put the 3'primer and 5'primer in separate tubes to check whether the primer itself form dimers during PCR. The result showed that the primer itself do not form dimer. Then i did the same but with both primers in a tube, and the 30-40bp possible primers amplified products was observed.
After that, i increased the annealing temperature from 50C to 62C. No possible primer dimers was observed in PCR with 62C annealing temperature. But at that high temperature, the RT-PCR surely didnt work as i have repeated several times.
I rechecked again n again on the Lau journal and Bartel and Ambros labs protocols. The primers i synthesized are exactly the same as the protocols above mentioned. Eventually, i come to this conclusion that, the quality of the primers is probable not good which result in this unknown pcr products. What is your opinion? I try synthesize new sets of primers from other company.
While waiting, i cut out the expected RT-PCR products (50-bp) from the gel and do re-amplification. Now i have done the concatamerization and trying to clone it. Hopefully everything will be fine.
As for the primers, I've realized too that they sometimes come of quite low quality. Yet, I used normal, unpurified primers for library amplification. Just the RT primer I ordered purified (HPLC, rnase free).
Dear Andrea,
My newly ordered primers had comed and i had used it for the miRNA amplication. The conditions of 30-40bp unknown pcr products persist.
Now, i start suspecting whether my reverse transcription successful or not. I used the superscript III, the protocol as described by the Bartel lab protocol. I used the RT primer as stated in the protocol.
I have been wondering why this protocol required to put in 100 pmole of RT primers while the Superscript III protocol just ask for less than 10 pmoles to start RT with the specific primer, in this case, the RT primers. So, is it possible the PCR product (30-40bp) i obtained is actually the artifacts of the excessive RT primers which somehow form dimers that enable it to be reverse-transcribed, thus led to bias during PCR and produced the 30-40bp PCR product? And this lead to the microRNAs cannot be RT-PCR? 
Or, the quantity of my ligated microRNAs is not enough to be RT-PCR?
Hope to hear you soon..
Thx a lot.
My newly ordered primers had comed and i had used it for the miRNA amplication. The conditions of 30-40bp unknown pcr products persist.
Now, i start suspecting whether my reverse transcription successful or not. I used the superscript III, the protocol as described by the Bartel lab protocol. I used the RT primer as stated in the protocol.
I have been wondering why this protocol required to put in 100 pmole of RT primers while the Superscript III protocol just ask for less than 10 pmoles to start RT with the specific primer, in this case, the RT primers. So, is it possible the PCR product (30-40bp) i obtained is actually the artifacts of the excessive RT primers which somehow form dimers that enable it to be reverse-transcribed, thus led to bias during PCR and produced the 30-40bp PCR product? And this lead to the microRNAs cannot be RT-PCR?
Or, the quantity of my ligated microRNAs is not enough to be RT-PCR?
Hope to hear you soon..
Thx a lot.
Hi Yew,
to make the library I used about 50 micrograms of home made low molecular weight rna, approximately equivalent to 500 micrograms of total rna. The limit was given by sample availability and rna quality. If the rna is not clean enough, and you load a lot on the gel, it may run irregularly and size selection will be affected.
Specificity in the RT is probably important, but I don't know if the conc of the RT primer is critical. Possibly yes, especially if the starting rna is low, and probably is important the purity of both RT primer and adapters. Perhaps there's a way to check your RT step. You could try to amplify a single miRNA known to be well expressed (there should be one, unless you're studing some very unusual organism), designing a primer spanning about half the miRNA and half the 5' adapter and using it combined with the 3' pcr primer. You would probably have to run a long pcr, like 40 cycles, or to re-amplify the first product, but I guess the result should be a single band.