i want to know what is better to use real time with sybr green otr taq man for quantification of miRNA
Hello Noha,
Both stains allow for the detection of nucleic acids, however there are a few key differences. SYBR green will nonspecifically stain any nucleic acid with the highest performance on double-stranded DNA and lower performance on single-stranded DNA and RNA. Taqman probes make use of the 5'-3' exonuclease activity of taq polymerase during polymerization. The taqman probe is a complementary DNA sequence that has a 5' fluorophore and a 3' quencher where the fluorescent signal from the fluorophore is silenced by the adjacent quencher until it gets cleaved during the PCR reaction to give a fluorescent signal. Since you want to quantify micro RNA I would suggest doing a reverse transcriptase PCR with real-time monitoring using a taqman probe or a molecular beacon. This will be more specific than SYBR green, however you will have to order a probe specific to each target and it will be more expensive.
A more economic method is to do the reverse transcriptase PCR and run your products through an agarose gel stained with SYBR green. If there is only one band, then the reaction is specific at the annealing temperature used and you can repeat the reaction on a real time monitoring platform. If there are multiple bands, all of the extra products would result in an overestimation of your product during real time monitoring because SYBR green will bind no specifically to all of them!
Good luck!
Alex
Hello Noha,
Both stains allow for the detection of nucleic acids, however there are a few key differences. SYBR green will nonspecifically stain any nucleic acid with the highest performance on double-stranded DNA and lower performance on single-stranded DNA and RNA. Taqman probes make use of the 5'-3' exonuclease activity of taq polymerase during polymerization. The taqman probe is a complementary DNA sequence that has a 5' fluorophore and a 3' quencher where the fluorescent signal from the fluorophore is silenced by the adjacent quencher until it gets cleaved during the PCR reaction to give a fluorescent signal. Since you want to quantify micro RNA I would suggest doing a reverse transcriptase PCR with real-time monitoring using a taqman probe or a molecular beacon. This will be more specific than SYBR green, however you will have to order a probe specific to each target and it will be more expensive.
A more economic method is to do the reverse transcriptase PCR and run your products through an agarose gel stained with SYBR green. If there is only one band, then the reaction is specific at the annealing temperature used and you can repeat the reaction on a real time monitoring platform. If there are multiple bands, all of the extra products would result in an overestimation of your product during real time monitoring because SYBR green will bind no specifically to all of them!
Good luck!
Alex
Hi Noha Said
Taqman is best method to quantify miRNA. The difficulty in the quantification of mature miRNA Is small size (Similar to the size of primers). As the advanced taqman miRNA reverse transcription kit contains stem loop primers for several miRNAs, it allows the conversion of all the small miRNA molecules into cDNA which is key step.
Hi Thomas Weppelmann,
you have given good explanation, Thank you,
As you have mentioned the economic method, is it possible to see the c-DNA of miRNA to visualise using agarose gels? I tried once but i couldn't see any bands after converting into c-DNA. If we opt for real time quantification using syber green, how can we design the primer for the c-DNA which is obtained by using taqman RT?.
Taqman is not the best. However, the probes are usually validated to work meaning it will amplify target template and not produce non-specific amplification. They do cost a lot though. LNA based probes (Exiqon) are also good but more expensive.
I have used SYBR-green method for amplifying low-template miRNA (NGS reads 30 Cts) from low copy templates which made data analysis diffcult at times. Instead of running gels you can try melt curve on the PCR machine to detect non-specific product. I used the nCode miRNA kit from invitrogen and just designed primers using online tools. With this apporach you can design primers for a lot of miRNA but hard to design primers for closely related sequences in a miRNA family i.e. Let-7.
"As you have mentioned the economic method, is it possible to see the c-DNA of miRNA to visualise using agarose gels?"
You may not be able to see the cDNA because maybe you are below the minimum concentration of DNA required for detection. Why are you checking for miRNA cDNA? The miRNA may not be a homogenous population with same sizes. Also what % is your agarose gel? What sizes of cDNA do you get with Taqman stem loops?
Hi Can Kiessling
Thankyou, for your reply,
I think u didn't follow the answers which is given by Thomas Weppelmann, i am just asking clarification for the alternative method mentioned in his answer.
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