Journal: Genome Research

Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

doi: 10.1101/gr.121426.111

Figure Lengend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

Article Snippet: Total RNA was isolated using TRIzol (Invitrogen), and small RNA samples were 5′ labeled by first treating with calf alkaline phosphatase (Roche) followed by phosphorylation with T4 polynucleotide kinase (NEB) and 32 P-γ-ATP.

Techniques: Labeling, Molecular Weight

Journal: Molecular Therapy. Nucleic Acids

Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

doi: 10.1016/j.omtn.2017.07.008

Figure Lengend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

Article Snippet: The protocol was modified as follows when CIP or T4 PNK treatment is necessary: (1) for CIP treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of CIP, 4 μL of 10× CutSmart buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min; and (2) for T4 PNK treatment, in 20 μL of products from one in vitro transcription reaction before DNase treatment, we added 1 μL of DNase (supplied with T7 Transcription Kit), 1 μL of T4 PNK, 4 μL of 10× T4 PNK buffer (NEB), and water to total volume of 40 μL, and incubated at 37°C for 15 min. All T7 in vitro transcription products were purified by Micro Bio-Spin P-30 Gel Columns, Tris Buffer, from Bio-Rad.

Techniques: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Nucleic Acids Research

Article Title: Identification of an hepatitis delta virus-like ribozyme at the mRNA 5?-end of the L1Tc retrotransposon from Trypanosoma cruzi

doi: 10.1093/nar/gkr478

Figure Lengend Snippet: Analysis of the 5′-hydroxyl nature of the ends of the cleavage 3′-products. Schematic cleavage reaction of the clone pGEM-T easy −61/L1Tc+77 RNA is represented in ( A ). The uncleaved RNA is expected to have 5′-triphosphate and 3′-hydroxyl ends. The cleavage 5′- and 3′-products are expected to have 2′,3′-cyclic phosphate and 5′-hydroxyl ends, respectively. The T4 polynucleotide kinase (T4 PNK) challenge is represented in ( B ). 5′-hydroxyl ends, not 5′-phosphate, are sensible to phosphorylation by T4 PNK. Same quantity of endogenously radiolabeled cleavage fragments was both ice preserved in reaction buffer and phosphorylated by T4 PNK using gamma 32 P-ATP as phosphate donor. The cleavage 3′-products of clones 7134, 55 and pGEM-T easy RNAs were further radiolabeled confirming the expected 5′-hydroxyl nature of their 5′-ends (solid arrowhead). The 61 nt in length RNA 5′-product of the cleavage of the pGEM-T easy construct is used as negative control in the phosphorylation reaction (the empty arrow indicates the labeled 5′-product). One of the 3′-products is pre-treated with alkaline phosphatase prior to being treated with T4 PNK. (marked with an asterisk).

Article Snippet: Experimental samples were 5′-end radiolabeled by phosphorylation reaction in 10 µl final volume for 20 min by adding 30 µCi γ32 P-ATP, 10 U T4 PNK enzyme (New England Biolabs) and the appropriate reaction buffer.

Techniques: Clone Assay, Construct, Negative Control, Labeling

Journal: bioRxiv

Article Title: tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

doi: 10.1101/2019.12.20.884650

Figure Lengend Snippet: Dynamic expression of placental/decidual microRNAs and tRNA fragments. (A) Scheme of sample collection for this figure. Placenta/decidua control samples were collected at embryonic development days E12.5, E13.5, E14.5 and E18.5 (corresponding to the 3 hours, 24 hours, 48 hours, 144 hours time point control samples). (B) Principle component analysis of microRNAs and tRFs from placenta/decidua control samples at different time points by sequence-level analysis. (C) Heatmap shows dynamic expression of placental/decidual microRNAs and tRFs across embryonic development time. Each row in heatmap represents one unique sequence. Expression values were calculated by log10RPM (reads per million total mapped reads) and averaged from 6 samples at each time point and then scaled across row. For visualization purpose, only abundantly expressed miRs or tRFs (mean expression cut-off 100 RPM) that show time-dependent changes are shown in the heatmap. A complete list of dynamically expressed miRs and tRFs please refer to Table S2. (D-E) Examples of dynamically expressed microRNAs. mmu-miR-215-5p (D) is up-regulated over time and mmu-miR-146b-5p (E) is down-regulated over time. (F-H) Examples of dynamically expressed tRFs, including down-regulated 5’ halves from tRNA GluTTC and tRNA GlyCCC . (D-F) Box plots showing RPM (reads per million total mapped reads) for specific miR or tRF sequence, with each dot represents one sample (n = 6 for each time point) and middle line represents median value. (G-H) qRT-PCR validation of temporal decrease of 5’ tRNA halves in both NT (no treatment control) and T4 PNK treatment samples. Error bars represent standard deviation from two biological replicates (male and female control samples pooled for each time point).

Article Snippet: T4 PNK treatment and RT-PCR 1 μg total RNA was treated with T4 PNK (NEB) in the presence of 10 mM ATP at 37 °C for 40 minutes and then cleaned up by ethanol precipitation.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Standard Deviation