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for improved profiling of transcripts strategies using technologies high-throughput sequencing
for improved profiling of transcripts strategies using technologies high-throughput sequencing
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机译：使用高通量测序技术改进转录本策略的分析
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摘要
Procedure for determining relative levels of transcription of a nucleotide sequence in muestrasde cDNA comprising the steps of: (a) Providing a first cDNA sample, (b) Perform a reduction reproducible complexity of the first cDNA sample 5 for unaprimera collection comprising the steps of: - digesting the cDNA with at least one restriction endonuclease to fragment it into fragments derestricción; - ligating the restriction fragments with at least one double-stranded synthetic oligonucleotide adapter quetenga one end compatible with one or both ends of the restriction fragments for producirfragmentos restriction linked to adapter; - contacting said ligated restriction fragments to adapter with one or more cebadoresoligonucleotídicos under hybridizing conditions, the said one or more primers oligonucleotídicosuna primer sequence which includes a section of nucleotide sequence complementary to part delal least one adapter and part of the remaining portion of the recognition sequence of restriction endonucleasade; y- amplifying said ligated restriction fragments by elongation of the adapter to one or more cebadoresoligonucleotídicos hybridized; (c) Mark the first collection for a first collection marked using at least unadaptador marked in step (b); (d) performing, consecutively or simultaneously, steps (a) and (b) with a second sample and / or subsequent unamuestra cDNA, using a different one for each cDNA sample mark for unasegunda collection marked and / or collection marked rear; (e) combining the first marked collection and the second labeled and / or marcadaposterior collection for a combined collection collection; (f) determining at least part of the nucleotide sequences of the collection marked mediantesecuenciación high performance; (g ) determine the frequency of the nucleotide sequence in the first sample and segundamuestra cDNA and / or later sample DNA; and (h) comparing the frequency of the nucleotide sequence in the first cDNA sample with the frequency dela nucleotide sequence in the second sample and / or a subsequent cDNA sample to obtain relative levels detranscripción of the nucleotide sequence in the samples cDNA.
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