Aim: To investigate the strand preference of the initial cleavage of human pre-miRNAs by human Dicer in vitro. Methods: We used a series of in vitro transcribed pre-miRNAs that were radioactively labeled at their 5′ or 3′ ends in cleavage reactions with recombinant human Dicer or HeLa cytoplasmic S100 extracts. Pre-miRNAs samples were purified by denaturing and native PAGE and only the stem-loop structures were used in the experiments. Products of cleavage reactions were resolved by denaturing PAGE, and scanned by phosphor-imaging. Results: Recombinant hDicer performs a biased first-cleavage in the pre-let-7b and hsa-pre-miR-17 3' strand. This result is recapitulated in HeLa S100 cytoplasmic extracts. Conclusion: The differential first-nick is observed in cleavage reactions only when stem-loops are substrates for hDicer.
With the advent of high-throughput sequencing of immunoglobulins (Ig-Seq), the understanding of antibody repertoires and its dynamics among individuals and populations has become and exiting area of research. There are an increasing number of computational tools that aid in every step of the immune repertoire characterization. However, since not all tools function identically, every pipeline has its unique rationale and capabilities, creating a rich blend of useful features that may appear intimidating for newcomer laboratories with the desire to plunge into immune repertoire analysis to expand and improve their research; hence, all pipeline strengths and differences may not seem evident. In this review we provide an organized list of the current set of computational tools, focusing on their most attractive features and differences in order to carry out the characterization of antibody repertoires so that the reader better decides a strategic approach for the experimental design, and computational analyses of immune repertoires.
Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP, were cloned into plasmids driven by RNA polymerase III promoter H1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.
The performance of local and global alignment algorithms circumscribe the alignment of sequences shorter than 30 nucleotides. Regardless of the computational approach applied, a series of diverse limitations accumulate as the length and similarity between the aligned sequences decreases, often resulting in alignment biases: local alignments have difficulties reporting correctly the number of matches and mismatches (m/mm) flanking the seed; global alignments lengthen the total alignment size and introduce gaps artificially. These biases compromise the accuracy of computational analysis of short sequences. Here we report ExtendAlign, a computational tool that overhauls and corrects the aforementioned bias generated by local and global alignments. ExtendAlign provides an end-to-end report of the accurate number of m/mm for all the nucleotides that flank a local alignment of short sequences, thus eliminating the artificial lengthening of the query size, the introduction of gaps, and the failure in reporting flanking m/mm. Since ExtendAlign combines the refinement and strength of global and local multiple sequence alignments, it delivers exceptional accuracy in correcting the alignment of dissimilar sequences in the range of ~35-50% similarity -also known as the twilight zone; indicating it can be adopted regularly whenever high accuracy is required for short-sequence alignments.
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