High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lncRNA (long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix (Xist antisense RNA), chicken LXN (latexin) and GFM1 (G elongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity of gene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 (TLR4) in Bama miniature pigs (Sus scrofa domestica) following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a nonclassical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 (RPL4) and TATA box-binding protein (TBP) were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear cells (PBMCs) during heat shock. The mRNA level of the TLR4 gene (both classical and spliced) in stressed pigs increased significantly (P<0.05). Further, the expression levels of the alternative spliced variant of TLR4 (TLR4-ASV) showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

A substantial fraction of miRNA* species are conserved in animals and can repress activities of target genes. This study aims to investigate the miRNA* species in duck skin by using Solexa sequencing. We obtained a total of 96 miRNA* species in two skin small RNA libraries and identified 56 miRNA/miRNA* (miR/miR*) pairs. Nucleotide bias of miRNA* indicated that the priority was C>A>U>G for the first nucleotide and U>C>A>G for the last nucleotide. Comparison analyses showed that 3´-U accounted for a higher proportion in the 56 miR/miR* pairs. Among the top 20 expressed miRNA* species, 17 were shared by two libraries and most of the miRNA* species were highly conservative, especially in the “seed region”. miR-199a* were expressed highly in our samples, which was also previously shown abundant in mouse hair follicle. Furthermore, four miRNA* species were predicted to target their genes in signal pathways of feather follicle development and feather morphogenesis despite very low levels.