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?
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You have something in Library Preparation and Sequencing.
Jordi
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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?
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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.
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I have a synthesize cDNA with random hexamers....
Please, send me your papers about this tema!
for: [email protected]
Tks so much!
Beta.
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