Only use a single internal control for normalization. These un-validated single BYL719 reference genes, however, were proved to be not always reliable under various experimental conditions. Therefore, more and more biologists pay their attention to the selection and validation of reliable reference gene expressed stably regardless of different experimental conditions from the species they are interested in, in order to avoid unnecessary errors in qRT-PCR analysis. Insecticide resistance is becoming a serious barrier for the sustainable control of pest insects. And the identification of insecticide resistance mechanisms would provide ways of detection and management of resistance. The qPCR has been extensively used to uncover the mechanisms of insecticide resistance, and it has been proven by a lot of publications that the overexpression of detoxifying enzymes and the reduced expression of insecticide target genes were responsible for insecticide resistance. Up to date, however, no universal and reliable reference genes were selected and evaluated in insects stressed with different classes of insecticides. In the present work, a total of eight candidate reference genes were validated in the tobacco whitefly treated with eight commonly used insecticides for the control of this pest. According to the final ranking order calculated by RefFinder, the five most stable reference genes for the eight tested insecticides treated whitefly were selected. Even for imidacloprid, acetamiprid and nitenpyram which belong to the same class of insecticides, the most stable reference genes were also different from each other. These results further proved that no single universal reference is available under different experiment conditions, and made the stability evaluation of reference gene necessary prior to the quantification of gene expression by qPCR. Very interestingly, the relative expression level of two detoxifying enzymes, Cyp6cm1 and GST from each of eight insecticides treated B. tabaci groups showed no significant difference between calculations using the selected reference gene set for each insecticide and calculations using the selected reference gene set for all eight insecticides. Combined with the results of Li et al that the expression of EF1a and GADPH was stable between the thiamethoxam susceptible and resistant B. tabaci strains, we recommended that our selected reference gene set can be used as reliable internal reference for the data normalization in qRT-PCR experiments using B. tabaci treated with different insecticides. Li et al suggested that 18SrRNA was stably expressed in B. tabaci when treated with thiamethoxam or under different temperatures. Based on the overall ranking by RefFinder, however, it was identified as the least stable reference gene in our study. When normalized with 18SrRNA, expressions of Cyp6cm1 and GST in B. tabaci treated with nitenpyram were significantly higher than those normalized with the set of most stable reference genes. These combined data strongly suggested the necessity of conducting customized reference gene selection for each and every experimental.

Of the mappable sequences, the majority of the sRNAs were 19–24 nt in size, which is typical of the sRNA of Dicer-processed products and similar to that of chicken and other fowl. In total, 1,328 known conserved miRNAs and 22 novel miRNAs were detected in goose ovary, which will greatly enrich the goose miRBase. In addition, we analyzed differential expression miRNA profiles between laying and broody ovary. The reads of these miRNAs sequences ranged from 1 to 3,085,441, indicating that Solexa sequencing can identify miRNAs with high and low expression. Therefore, Solexa sequencing is a more accurate and efficient approach for studying sRNAs than the traditional cloning method, which only identified 23 miRNAs. Some miRNAs were detected in only one sRNA library, such as miR-34 and miR-129, and some miRNAs showed significantly different expression between the two libraries, such as miR-146 and miR-202, indicating that these miRNAs may have physiological functions in goose ovary tissue. Because the identification of miRNA candidates was based on the chicken genome sequences, there may be a few sequence differences in the goose. A total of five conserved miRNAs were randomly selected for RTqPCR. Four conserved miRNAs were validated; one could not be detected by qRT-PCR, possibly because of inappropriate primer design, very low expression, or because it is a false-positive result, and requires further experimental verification. Of the four validated miRNAs, miR-320 has been extensively studied in the ovary. The expression of miR-320 is increased in the ovary of rats with polycystic ovary syndrome, and was also found to be significantly up-regulated in TGF-b1- stimulated mouse ovary preantral granulosa cells. This indicates that miR-320 may participate in ovarian function. In addition, miR-202 and miR146 were proven to be associated with reproductive hormone secretion. A large number of studies have shown that miR-143 might be involved in mammalian reproductive activities. In this study, we found abundant expression of miR-143, which was represented by 185,110 and 283,032 reads in the BO and LO libraries, respectively. However, miR-143 did not show significant differential expression between LO and BO although miR-143*, a member of the miR-143 family, did. Because no 3’UTR database is available it is difficult to predict targets of goose miRNAs. To provide further insight into the physiological functions of miRNAs in goose ovary function, the presumed target genes for the differentially expressed miRNAs were predicted by aligning miRNA sequences to the goose transcriptome. Analysis by GO and KEGG showed that the putative target genes appear to be involved in hormone secretion and reproduction process. These results indicated that some miRNAs might be involved in ovary cell proliferation, apoptosis, and differentiation. Although a large number of target gene candidates were predicted using bioinformatics tools, validation of the relationship between miRNAs and mRNA transcripts requires further experimental WZ4002 evidence.