Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

The effects of a globin blocker on the resolution of 3'mRNA sequencing data in porcine blood.

BACKGROUND: Gene expression profiling in blood is a potential source of biomarkers to evaluate or predict phenotypic differences between pigs but is expensive and inefficient because of the high abundance of globin mRNA in porcine blood. These limitations can be overcome by the use of QuantSeq 3'mRNA sequencing (QuantSeq) combined with a method to deplete or block the processing of globin mRNA prior to or during library construction. Here, we validated the effectiveness of QuantSeq using a novel specific globin blocker (GB) that is included in the library preparation step of QuantSeq. RESULTS: In data set 1, four concentrations of the GB were applied to RNA samples from two pigs. The GB significantly reduced the proportion of globin reads compared to non-GB (NGB) samples (P = 0.005) and increased the number of detectable non-globin genes. The highest evaluated concentration (C1) of the GB resulted in the largest reduction of globin reads compared to the NGB (from 56.4 to 10.1%). The second highest concentration C2, which showed very similar globin depletion rates (12%) as C1 but a better correlation of the expression of non-globin genes between NGB and GB (r = 0.98), allowed the expression of an additional 1295 non-globin genes to be detected, although 40 genes that were detected in the NGB sample (at a low level) were not present in the GB library. Concentration C2 was applied in the rest of the study. In data set 2, the distribution of the percentage of globin reads for NGB (n = 184) and GB (n = 189) samples clearly showed the effects of the GB on reducing globin reads, in particular for HBB, similar to results from data set 1. Data set 3 (n = 84) revealed that the proportion of globin reads that remained in GB samples was significantly and positively correlated with the reticulocyte count in the original blood sample (P < 0.001). CONCLUSIONS: The effect of the GB on reducing the proportion of globin reads in porcine blood QuantSeq was demonstrated in three data sets. In addition to increasing the efficiency of sequencing non-globin mRNA, the GB for QuantSeq has an advantage that it does not require an additional step prior to or during library creation. Therefore, the GB is a useful tool in the quantification of whole gene expression profiles in porcine blood.

A live zebrafish-based screening system for human nuclear receptor ligand and cofactor discovery.

Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors.

Expression profiling of vitamin D treated primary human keratinocytes.

Vitamin D has attracted much attention by its ability to stop cell proliferation and induce differentiation, which became of particular interest for the treatment of cancer and psoriasis. We performed an expression profile of 12 hours and 24 hours 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) treated primary human keratinocytes, to determine the changes in gene expression induced by the steroid in order to improve our understanding of the biological activity of 1alpha,25(OH)(2)D(3). This we expect to be useful for establishing a test system for vitamin D analogs or might open new therapeutic targets or uses for the hormone. For the filter array experiments a non-redundant set of 2135 sequence verified EST clones was used. The normalized raw data of 2 filters per time point were combined and subjected to SAM analysis to further increase the statistical significance. 86 positive and 50 negative genes were identified after 12 h. The numbers went down to 43 positive and 1 negative gene after 24 h of treatment. Fifteen genes are up-regulated over a longer period of time (12 h and 24 h). Results were verified by real-time PCR and/or Northern blots. Targets identified are involved in intracellular signaling, transcription, cell cycle, metabolism, cellular growth, constitution of the extracellular matrix or the cytoskeleton and apoptosis, immune responses, and DNA repair, respectively. Expression profiles showed an initial stop of proliferation and induction of differentiation, and resumed proliferation after prolonged incubation, most likely due to degradation of the hormone.

Expression profiling of aging in the human skin.

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms (C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3-4 years of age) and five old (68-72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

Optimized RNA amplification using T7-RNA-polymerase based in vitro transcription.

The use of expression profiling to explore a cell's transcriptional landscape has exploded in recent years. In many cases, however, the very limited amount of starting material poses a major problem, making the amplification of the isolated RNA obligatory. The most prominent amplification method used was developed by the Eberwine lab in 1990: cDNA synthesis is started with an oligo(dT) primer containing a T7 RNA polymerase promoter. After second-strand synthesis RNA is transcribed in vitro using T7 RNA polymerase. It has been demonstrated that antisense RNA amplification not only preserves the fidelity of RNA-based microarray analysis but even improves the sensitivity. In our aim to improve the yield of in vitro transcription reactions and to facilitate the use of amplified RNA for the construction of cDNA libraries we tested a series of T7 primers with different 3' flanking sequences containing restriction sites. In addition we tested the impact of different DNA polymerases used for synthesizing the templates on the efficiency of the in vitro transcription reaction. A total of 28 different oligo(dT)-T7 promoter primers were tested. Two of them showed a dramatically increased yield of RNA from the in vitro transcription reaction. The combination of the improved second-strand synthesis with the new T7 primer increased the RNA yield 60-fold compared to the yield of standard procedures.

Identification of vitamin D target genes in human keratinocytes by subtractive screening.

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)) imposes cell cycle block in late G1 phase in cultured human keratinocytes. We wanted to identify early vitamin D target genes using a subtractive screening approach. Human foreskin keratinocytes were grown to about 70% confluence, treated with 2 x 10(-7) M 1alpha,25(OH)(2) D(3) or left untreated and RNA from both populations were isolated after 22h of incubation. cDNA was synthesised and cloned into plasmid vectors. For screening of the libraries, cDNA was amplified in vitro using T7 RNA polymerase and then the amplified RNA (driver, control population) and single stranded cDNA (tester) were used for subtractive hybridisation. Heterohybrids were then separated from single stranded nucleotides using a hydroxyapatite column. The radiolabeled single stranded cDNA was used for screening a colony blot. Positive clones were rescreened, plasmid DNA was isolated and used for verifying the results by Northern blot analysis, using RNA isolated from untreated keratinocytes, as well as RNA isolated after 6h, 12h and 24h of vitamin D treatment.