Poly(A) Tail treatment?

[betacamp]

I need make a brainstorm about possible protocols for treatment of poly(A) Tail in the case of direct RNA or cDNA sequencing for use in 454. Somebody has some tip?

[jordi]

https://www.454.com/downloads/protocols/Transcriptome_NatureMethods.pdf

You have something in Library Preparation and Sequencing.
Jordi

[betacamp]

Tks Jordi!
I Look in Annual meeting of the Society for Molecular Biology and Evolution/Barcelona 2008 and dont have a manuscript or paper about?

[AKroxy]

which polyA treatment best?

In the link posted, there are a few ways to treat the polyA tail - interrupt it, cut it off, or synthesize cDNA with random hexamers. All the publications I've seen have used the interrupted polyT primer, but a short email I got from someone at Roche suggested that random hexamers or cutting off the tail would be better, especially with titanium.
Has anyone here used one of these other methods?

[wiggy]

We are using random ninemers after poly A purification. We are seeing large 2-3 KB transcripts, some transcribed at low levels, making it into the first strand using this method. Our qRTPCR primers are designed near the 5' terminus of these mRNA's.

This is mRNA that has been run twice through Omega's polyA purification kit.

Oligo dt priming is not really necessary in my opinion, nor is the smart protocol for cDNA synthesis. Oligo DT is good for non purified RNA, but there is a good chance that one random primer will bind near the 3' end.

Smart is good for sequencing entire full length transcripts, but in massively parallel applications like 454, where the transcripts will just be split any way, I can't see the point in expensive smart technology.

By the way, if anyone knows a procedure to quantify the amount of first strand cDNA converted into second strand I would really appreciate it.

[AKroxy]

Thanks Wiggy for your help!
I couldn't see any good reason to use smart over other methods to synthesize ds cDNA, but every one seems to use it - so I wondered if I was missing something!

[betacamp]

I have a synthesize cDNA with random hexamers....

Please, send me your papers about this tema!


for: [email protected]

Tks so much!

Beta.