5 replies to this topic

[rhizorick]

I have completed a qRT-PCR experiment examining expression of 2 miRNAs in Medicago truncatula.  I have ran the PCR products on gels and got single bands around 90 bp for each miRNA.  90 bp bands would be expected because I used the stem-loop method of reverse transcribing which adds length to the original 21 nt sequence.  Now I want to sequence the PCR products, but so far I've tried 2 facilities and both of them failed to give me good results.

Has anyone here ever tried to sequence a miRNA amplicon from a real-time Rev. Trans-PCR experiment?

If so, please let me know how.

Thanks,
Rick

[pcrman]

I am not sure if the dye in the qPCR mix interfere with sequencing reaction. Why don't you amplify the product by regular RT-PCR. Make sure you get sufficient product for sequencing.

[Fizban]

pcrman, on 20 April 2011 - 06:28 PM, said:

I am not sure if the dye in the qPCR mix interfere with sequencing reaction. Why don't you amplify the product by regular RT-PCR. Make sure you get sufficient product for sequencing.

I'm far from my sequencing times but as far as i remember sequencing by dideoxy method gives good results 30-40 nts downstream primer binding site so that you'd loose a lot of info. stem loop miRNAs amplification should be devoid of dyes and you shold have clean DNA if you did gel elution of your bands.
back to the topic my guess is that ligation into a sequencing  vector'd help a lot
let us know
Fiz

[rhizorick]

pcrman, on 20 April 2011 - 06:28 PM, said:

I am not sure if the dye in the qPCR mix interfere with sequencing reaction. Why don't you amplify the product by regular RT-PCR. Make sure you get sufficient product for sequencing.

The products are first ran on a 2% agarose gel and then extracted using a Qiagen kit.  Theoretically the Sybr green mix will be separated out by the gel and then residual should be washed away by the kit (there are 2 washing steps).

However, you might be on to something with the end-point PCR.  Perhaps I can get greater amplification and more product.  

Thanks

[rhizorick]

Fizban, on 21 April 2011 - 04:22 AM, said:

pcrman, on 20 April 2011 - 06:28 PM, said:

I am not sure if the dye in the qPCR mix interfere with sequencing reaction. Why don't you amplify the product by regular RT-PCR. Make sure you get sufficient product for sequencing.

I'm far from my sequencing times but as far as i remember sequencing by dideoxy method gives good results 30-40 nts downstream primer binding site so that you'd loose a lot of info. stem loop miRNAs amplification should be devoid of dyes and you shold have clean DNA if you did gel elution of your bands.
back to the topic my guess is that ligation into a sequencing  vector'd help a lot
let us know
Fiz

I've never really done ligation into a vector, but I've hear that it can be difficult to clone a 90 bp product.  Anyone know much about this?

[rye]

Sequence of loop RT-PCR amplicon for miRNA seems to be provided by primer and adaptor, therefore, I wonder the purposes of such sequencing.
Here is a reference for different approach of sequencing small RNAs amplicons.
http://www.springerl...28/fulltext.pdf

rhizorick, on 20 April 2011 - 01:05 PM, said:

I have completed a qRT-PCR experiment examining expression of 2 miRNAs in Medicago truncatula.  I have ran the PCR products on gels and got single bands around 90 bp for each miRNA.  90 bp bands would be expected because I used the stem-loop method of reverse transcribing which adds length to the original 21 nt sequence.  Now I want to sequence the PCR products, but so far I've tried 2 facilities and both of them failed to give me good results.

Has anyone here ever tried to sequence a miRNA amplicon from a real-time Rev. Trans-PCR experiment?

If so, please let me know how.

Thanks,
Rick

Edited by rye, 08 May 2011 - 03:47 PM.