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A final test of the generated model was
To confirm the data obtained from high-throughput sequencing, qRT-PCR was carried out to validate the ICG001 patterns of cold-responsive and new miRNAs (Fig. 3). Some miRNAs within the same family share the same mature miRNA sequences but have different precursors; therefore, they are barely distinguishable by qRT-PCR. Consequently, in qRT-PCR analyses, pto-miR167c–d, pto-miR169f–i, pto-miR171i–k, and pto-miR395b–k could represent 19 different miRNAs. The cold-responsive miRNAs (pto-miR167c–d, pto-miR169f–i, pto-miR171i–k, and pto-miR395b–k) showed the same trends as that predicted by Solexa sequencing in response to the 8-h cold treatment. Transcripts of pto-miR167c–d were up-regulated and peaked at 2 h, while those of pto-miR169f–i were down-regulated and peaked at 8 h. Expression of pto-miR171i–k peaked at 8 h, then decreased sharply and remained similar levels at 14 and 20 h of cold stress. Expression of pto-miR395b-k was down-regulated at 2, 8, and 20 h, but up-regulated at 14 h of cold stress. Meanwhile, two new miRNAs and their corresponding miRNA?s were validated by qRT-PCR. The results revealed that the relative expression trends of new miRNAs and miRNA?s were opposite; that is, when the expression level of pto-miRS16 increased steadily, that of pto-miRS16? deceased. Similarly, the increase in pto-miRS6 was accompanied by a decrease in pto-miRS6?. Pto-miRS6 was expressed at far greater levels than pto-miRS6?, while pto-miRS16? was expressed at markedly higher levels than pto-miRS16.





 
 
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