title: Illumina HiSeq 2000 sequencing; miRNA sequencing of pheochromocytomas (172 samples)
design: miRNA sequencing of pheochromocytomas (172 samples)
library_construction_protocol: miRNA-Seq libraries are performed from at least 1ug of extracted total RNA with a RIN greater than 7. Before starting, total RNAs are purified with miRNeasy kit wich allows the selection of the small RNA fraction less than 100b. first, a 3' adenylated DNA adaptor is ligated to the enriched sample in the absence of ATP preventing the self-ligation of miRNAs.Then a 5' RNA adaptor is ligated in the presence of ATP at the other end of the miRNAs.The RT primer complementary of the 3' adaptor is added at this stage with which it will form a duplex thereby reducing the ligation between adaptors.A reverse transcription is therefore perfomed from the RT primer and finally these captured miRNAs are amplified by PCR with primers complementary to the 3' and 5' adaptors.During this PCR a specific barcode is incorporated allowing individualisation of each library.Each PCR is loaded on the Fragment Analyzer (AATI) for a precise quantification of each miRNA peak of interest.Based on these results a equimolar pool of about ten of different samples are performed.Finally the pooled PCR product is loaded on PAGE in order to excise the band of miRNA that is extracted and purified on a Qiagen MinElute column.Finally the pooled PCR product is loaded on PAGE in order to excise the band of miRNA that is extracted and purified on a Qiagen MinElute column.
attributes.tag: Experimental Factor: overall survival event
attributes.value: 0
attributes.tag: Experimental Factor: overall survival delay
attributes.value: 7 month