To solve these problems, and in order to identify the most reliable oligos for a definitive selleck turbot microarray, a pilot microarray was developed. In this pilot microarray, oligos were designed both in forward and reverse sequence orientation. In addition, several filtration criteria were followed to analyze microarray data. This strategy allows, on one hand, to identify the sense strand of the non annotated sequences, but also to identify false annotation of genes. On the other hand, this procedure also allows studying the frequency of putative natural antisense tran scripts in turbot transcriptome. The importance of NATs, which can regulate eukaryotic gene expression, has emerged in the last decade.
A NAT is a single stranded RNA sequence complementary to messenger RNA and includes various classes of short RNAs including micro RNAs, promoter associated transcripts and long non protein coding RNAs. The amount of NATs in eukaryotic cells remains unclear. It had been reported that over 20% of human transcripts might form sense antisense pairs, but large scale Inhibitors,Modulators,Libraries cDNA sequencing suggested that antisense transcription is more common than previously thought. Recently, it has been shown that up to 72% of the transcripts had antisense partners in human and mouse transcriptomes. High throughput sequencing strategies have revealed a plethora of non protein coding transcripts from both genic and intergenic regions. Data on miRNAs, one of the short NAT classes, has been already published in rainbow trout and halibut Hippoglussus hippoglossus.
Due to their increasing importance, the study of NATs cannot be longer ignored in transcriptome studies. The functionality of the oligos Inhibitors,Modulators,Libraries included in the pilot microarray was checked by hybridizing the same RNA used for the Sanger and 454 sequencing strategies. To analyze microarray data two filtration criteria were applied. Once the first filtration process Inhibitors,Modulators,Libraries was com pleted 37,759 signals in forward and 33,489 in reverse oligos still remained. Then, a second fil tration with two additional filtering criteria was performed to select the best performing oligo probes. As seen after this additional filtration process, among the 94,582 probes there were 53,534 with no signal. After the two rounds of filtration, a total of Inhibitors,Modulators,Libraries 41,048 remaining oligos yielded signal in at least one tissue or in both.
Inhibitors,Modulators,Libraries As a result of this filtration strategy, the remaining oligos were se lected to be included in the updated turbot microarray. In the development of a custom microarray for the European sea bass, a similar strategy was followed to study NATs ex pression. Although a lesser amount of sequences was designed for this purpose, identification of NATs was also achieved. It is remarkable that after Brefeldin the second filtration, 2,976 sequences still showed signal in both strands in both types of tissues.