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  • 99
    New England Biolabs nebnext poly a mrna magnetic isolation module
    Electropherograms from five embryos of soybean seeds harvested in 1994 (stored 23 years, orange dashed lines) or 2015 (stored 2 years, green solid lines). (A) Total RNA from each sample. The differences in peak heights in 1994H relative to 2015H samples indicate RNA degradation. (B) DNAse-treated, <t>poly-A</t> RNA. The most abundant mRNAs are smaller in 1994H seeds than 2015H seeds.
    Nebnext Poly A Mrna Magnetic Isolation Module, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 3210 article reviews
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    nebnext poly a mrna magnetic isolation module - by Bioz Stars, 2020-09
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    99
    Thermo Fisher dynabeads mrna purification kit
    Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the <t>antibody-ribosome-mRNA</t> complex with protein A <t>dynabeads.</t> After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.
    Dynabeads Mrna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Exosome Diagnostics mrnas
    Validated miRNA targets and pathways overlap with sporadic YOAD transcriptome changes. a Five-set Venn diagram showing 2899 <t>mRNAs</t> identified as differentially expressed in the posterior cingulate cortex (PCC) overlap of published microarray data (GSE39420). A total of 874 miRNA targets altered in the exosomes of CSF from sporadic YOAD patients overlapped with the entire microarray dataset. b KEGG pathways were identified using DAVID (v6.8) with the 874 overlapping validated miRNA targets altered in CSF-derived exosomes from sporadic YOAD. Enriched KEGG pathways ( y -axis) represented as −log 10 ( p value) ( x -axis). The number of genes shared for each pathway is shown at the end of each pathway bar
    Mrnas, supplied by Exosome Diagnostics, used in various techniques. Bioz Stars score: 92/100, based on 1093 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq stranded mrna library prep kit
    gas6 may only have subtle roles in caudal hindbrain development. a WT and gas6 mutant embryos were assayed for expression of valentino (r5/r6) (i), hoxb3a (r5-spinal cord) (ii), hoxa3 (r5/r6) (iii), islet1 (cranial nerves)(iv) and dm20 (oligodendrocyte marker) (vi) by ISH, as well as for the presence of OPCs and abducens neurons by crossing to the Tg (olig2:EGFP) vu12 line (v). In column (iv), yellow brackets mark cranial nerve V, blue brackets mark cranial nerve VII and red brackets mark cranial nerve X. White brackets indicate the presence of abducens (cranial nerve VI) in column (v). b Schemes showing RNA-seq library synthesis. Hindbrain tissue was dissected from 48 hpf gas6 mutant embryos in the olig2:eGFP background. Total RNA was collected from pools of hindbrain tissue and was used in library synthesis following the <t>TruSeq</t> Stranded <t>mRNA</t> Library Prep Kit (Illumina) protocol. c 1590 differentially expressed genes were identified from RNA-Seq where 41 out of the 928 up-regulated genes and 78 out of the 662 down-regulated genes were expressed in the hindbrain. GO terms related to Biological Processes were identified in both up-regulated and down-regulated genes using DAVID. d A subset of differentially expressed genes was validated via qPCR from independently collected hindbrain tissue samples. e ISH analysis of representative differentially expressed hindbrain genes (i) neurod6b , (ii) atoh1b and (iii) olig4 show no detectable change in expression pattern in gas6 mutant embryos
    Truseq Stranded Mrna Library Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 3752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/truseq stranded mrna library prep kit/product/Illumina Inc
    Average 99 stars, based on 3752 article reviews
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    99
    Millipore mrna
    Mutations in SNU13 inhibit <t>pre-mRNA</t> splicing. ( A ) Primer extension analysis of pre-U3 snoRNA splicing. Strain YGALSNU13 was grown in the presence of plasmid-based wild-type SNU13 or mutants E59A, R84A/V81L, or Δ111-126. Total <t>RNA</t> was harvested
    Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads mrna direct kit
    Distinct nucleotide composition patterns at <t>mRNA</t> 3′-end regions. Nucleotide composition plot of poly(A) site, −50 to +50 window for <t>Direct</t> RNA sequencing data from human liver RNA sample in A ) and 3Seq data from human U2OS cell line in B ). ( C ) 3Seq metagene analysis of −150 to +100 window centered at the cleavage site. The percent reads are calculated by dividing the read coverage on each position by all the number of reads mapped to this window. 3Seq reads are significantly enriched in the −100 to 0 position, with dramatic drop downstream from the 0 position. ( D ) Percent reads plot of 3′ ends of trimmed reads centered at the cleavage site whose +1 position is T, G, or C. ( E ) H3K36me3 metagene plot centered at the cleavage site. Distal peaks (blue) are defined as the most 3′ peak assigned to a given gene. Proximal peaks (red) are peaks mapped to RefSeq and Ensembl-annotated that are proximal in location to the distal peaks.
    Dynabeads Mrna Direct Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher mrna
    Transfection of non-phagocytic cells, using <t>mRNA:LPNs</t> and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p
    Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 89710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad mrna levels
    Effects of intrathecal ATL on the expression of <t>IL-1β,</t> IL-6 and TNF-α <t>mRNA</t> assessed by RT-PCR in the spinal cords of rats with CIBP. The L4-L5 spinal cords were used to test 2 hours after i.t. injection with ATL (0.3 nmol/20 μl) or NS (20 μl) on day 11 after surgery. Data are expressed as means ± SEM. ** P
    Mrna Levels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 4144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligotex mrna mini kit
    The novel RNA species is devoid of a poly-A tail dstet5 cells were treated with 10 μM LAM or 0.1 μM ETV for 3 days in the absence of tet. Total cellular RNA were extracted with TRIzol reagents and polyA + <t>mRNA</t> were extracted with an <t>Oligotex</t> mRNA mini kit from total RNA preparations. Each of the lanes was loaded with 6 μg of total cellular RNA or polyA-selected RNA from 6 μg of total cellular RNA from dstet5 cells left untreated or treated with the indicated concentrations of viral DNA polymerase inhibitors. To ensure each of the lanes are loaded with equal amount of RNA, each of the polyA-selected RNA samples was mixed with 6 μg of total cellular RNA prepared from parental LMH cells. DHBV RNA were detected by Northern blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.
    Oligotex Mrna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligotex mrna mini kit/product/Qiagen
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    94
    Illumina Inc mrna seq
    Experimental design and construction of the pAs reference Study design. 3′ READS method was used to identify the pAs expressed in the fibroblasts from C57BL/6J and SPRET/EiJ mouse strains. 3′ <t>mRNA‐Seq</t> method was used to quantify the usage of the identified pAs in both two parental strains and their <t>F1</t> hybrids. The frequencies of 13 known PAS motifs in the 100 nt upstream region of pAs identified in C57BL/6J. pseudo_pAs represents pAs identified by using reads without non‐genomic T ( Materials and Methods ). raw_pAs and filtered_pAs represent the pAs determined by the PASS reads with and without further filtering, respectively ( Materials and Methods ). X ‐axis shows different types of PAS motifs, and y ‐axis shows the percentage of pAs with the specific motif in the upstream region. See Fig EV1 C for the results from SPRET/EiJ. 13,406 pAs identified in this study were located within 50 nt away from the 3′ end of RNA transcripts annotated in Ensembl. X ‐axis shows the distance between the 13,406 pAs identified in this study and the annotated pAs. Y ‐axis represents the density. Inset: Barplot shows the number of pAs with representative cleavage site identical to the annotated transcript ends as well as those within 5 nt upstream or downstream to the annotated ends, respectively. The pie chart shows the percentage of protein‐coding genes with different number of pAs. Schematic definition for different types of pAs. See Materials and Methods for the details. Pie chart shows the percentage of different types of pAs identified in this study.
    Mrna Seq, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 2649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc mrnas
    Pathway-based gene set enrichment analyses of differentially expressed <t>mRNAs</t> or <t>miRNAs.</t> Protein-coding genes were ranked according to the number of pathways in which they were prioritized as core genes (most significantly deregulated genes). miRNAs were ranked according to the number of pathways in which their target genes were prioritized as core genes. Columns in green/red represent the genes or miRNAs that were down/upregulated on average in at least two ccRCCs respectively; columns in black represent the genes or miRNA targets involved in at least one cancer-associated pathway. The height of the columns in different colors represents the number of pathways where the genes or miRNA targets were ranked as core genes. A: Genes ranked in the top 20 based on the results of pathway-based gene set enrichment analysis. B: miRNAs ranked the top 20 based on the results of pathway-based gene set enrichment analysis of their target genes.
    Mrnas, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 1722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad mrnas
    Expression levels of FSHβ (A, B) and LHβ (C, <t>D)mRNAs</t> in Nile tilapia pituitaries cultured with different concentrations of NKB (A, C) or NKBRP (B, D) peptides. Relative abundance of the mRNAs was normalized to the amount of <t>GAPDH</t> by the comparative threshold cycle method using qRTPCR. Results are means ± SEM (n=5–8). * indicates significant difference from 0 μM group (P
    Mrnas, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1617 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mrnas/product/Bio-Rad
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    99
    Thermo Fisher microbexpress bacterial mrna enrichment kit
    Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion <t>MICROBExpress™</t> Bacterial <t>mRNA</t> Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
    Microbexpress Bacterial Mrna Enrichment Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dynabeads mrna direct micro kit
    The procedure for the isolation of antipodal cells, <t>mRNA</t> extraction and cDNA amplification. Briefly, the ovules are dissected from flowers at the stage 12c and are further dissected by handmade razor under fluorescent microscope to obtain ovule fragments containing antipodal cells. The antipodal cells are further separated from other tissues by microneedles. The isolated antipodal cells are transferred into the 2x lysis buffer. <t>Dynabeads</t> are used for mRNA extraction and SMARTer Ultra Low Input RNA kit for Sequencing-v3 is used for cDNA amplification subsequently.
    Dynabeads Mrna Direct Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Electropherograms from five embryos of soybean seeds harvested in 1994 (stored 23 years, orange dashed lines) or 2015 (stored 2 years, green solid lines). (A) Total RNA from each sample. The differences in peak heights in 1994H relative to 2015H samples indicate RNA degradation. (B) DNAse-treated, poly-A RNA. The most abundant mRNAs are smaller in 1994H seeds than 2015H seeds.

    Journal: Journal of Experimental Botany

    Article Title: Exploring the fate of mRNA in aging seeds: protection, destruction, or slow decay?

    doi: 10.1093/jxb/ery215

    Figure Lengend Snippet: Electropherograms from five embryos of soybean seeds harvested in 1994 (stored 23 years, orange dashed lines) or 2015 (stored 2 years, green solid lines). (A) Total RNA from each sample. The differences in peak heights in 1994H relative to 2015H samples indicate RNA degradation. (B) DNAse-treated, poly-A RNA. The most abundant mRNAs are smaller in 1994H seeds than 2015H seeds.

    Article Snippet: The NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA) was used to enrich for mRNA according to the manufacturer’s protocol.

    Techniques:

    The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: The KH domains of IGF2BPs are critical for m 6 A recognition and binding ( a ) Schematic structures showing RNA binding domains within IGF2BP proteins and a summary of IGF2BP variants used in this study. Blue boxes are RRM domains, red boxes are wild-type KH domains with GxxG core, and grey boxes are inactive KH domain with GxxG to GEEG conversions. ( b ) RNA pulldown followed by Western blotting showed in vitro binding of ssRNA baits with wild-type (wt) or KH domain-mutated IGF2BP variants, representative of 3 independent experiments. ( c ) In vitro binding of CRD1 RNA probes with wild-type or KH3-4 mutated IGF2BPs, representative of 3 independent experiments. ( d ) The association of wild-type and KH3-4 mutated IGF2BPs with MYC CRD in HEK293T cells as assessed by RIP-qPCR. ( e ) Relative luciferase activity of CRD reporters in HEK293T cells with forced expression of wild-type or mutated IGF2BP2 variants. ( f ) Changes in MYC mRNA levels in Hela cells with empty vector or forced expression of wild-type or KH3-4 mutated IGF2BPs one hour post-heat shock (HS). Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in d , e , f (*, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, RNA Binding Assay, Western Blot, In Vitro, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Two Tailed Test

    IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: IGF2BPs regulate MYC expression through binding to methylated CRD ( a ) Distribution of m 6 A peaks across MYC mRNA transcript. The coding region instability determinant (CRD) region is highlighted in yellow. m 6 A-seq was repeated twice while RIP-seq was performed once.( b ) RIP-qPCR showing the association of MYC CRD with FLAG-tagged IGF2BPs in HEK293T cells. ( c ) Enrichment of m 6 A modification in MYC CRD as detected by gene specific m 6 A qPCR assay. ( d ) RIP-qPCR showing the binding of METTL3 and METTL14 to the MYC CRD. ( e ) RNA pulldown of endogenous IGF2BP proteins from HEK293T nuclear extract using synthetic CRD RNA fragments, CRD1 and CRD2, with (m 6 A) or without (A) m 6 A modifications. Images are representative of 3 independent experiments. ( f ) Relative firefly luciferase (Fluc) activity (i.e., protein level; left) and Fluc mRNA level (right) of wild-type (CRD-wt) or mutated (CRD-mut) CRD reporters in HEK293T cells with ectopically expressed IGF2BP1, IGF2BP2, or IGF2BP3. ( g ) RIP-qPCR detecting the in vivo binding of Flag-IGF2BPs to the transcripts of CRD-wt or CRD-mut luciferase reporter in HEK293T cells. ( h and i ) Relative luciferase activity of CRD-wt or CRD-mut in Hela cells with or without stable knockdown of IGF2BPs (h) or METTL14 (i). ( j ) Relative luciferase activity of CRD-wt or CRD-mut in METTL14 stable knockdown or control Hela cells with ectopic expression of IGF2BPs . For all luciferase assays, the Fluc/Rluc ratio (representing luciferase activity) of CRD-wt with empty vector or shNS was used for normalization. Values are mean±s.d. of n =3 independent experiments, and two-tailed Student’s t -tests were used in b , c , d , f , g , h , i , j . (**, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Expressing, Binding Assay, Methylation, Real-time Polymerase Chain Reaction, Modification, Luciferase, Activity Assay, In Vivo, Plasmid Preparation, Two Tailed Test

    Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Journal: Nature cell biology

    Article Title: Recognition of RNA N6-methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation

    doi: 10.1038/s41556-018-0045-z

    Figure Lengend Snippet: Selective binding of IGF2BPs to m 6 A-modified RNAs ( a ) Identification of m 6 A specific binding proteins by RNA affinity chromatography using ssRNA probes with methylated (red) or unmethylated (green) adenosine. Silver staining (lower left) and Western blotting (lower right) showed selective pulldown of ~68kDa IGF2BP proteins from HEK293T nuclear extract. Western blot images were representative of 3 independent experiments. ( b ) Enrichment of m 6 A consensus sequence “GGAC” in the binding sites of RBPs. The three IGF2BP paralogues were shown in red, while the YTH domain proteins were shown in orange. ( c ) Quantification of m 6 A/A and m 6 A/AGCU ratios by LC-MS/MS in RNAs bound by ectopically expressed IGF2BP1 (chicken ZBP1), IGF2BP2 (human), or IGF2BP3 (human). Values are mean of n =2 independent experiments and individual data points are showed. ( d ) Overlap of IGF2BP target genes identified by RIP-seq and published PAR-CLIP in HEK293T cells. RIP-seq was performed once. P value was calculated by Fisher’s test. ( e ) Venn diagram showing the numbers of shared high-confidence targets ( i.e. , CLIP+RIP targets) amongst IGF2BP paralogues. P value was calculated by Fisher’s test. ( f ) Top consensus sequences of IGF2BP binding sites and the m 6 A motif detected by HOMER Motif analysis with PAR-CLIP data. ( g ) Pie charts showing numbers and percentages of IGF2BP high-confidence target genes that contain m 6 A peaks. The m 6 A-seq data was reported in Ref. 3 . ( h ) Metagene profiles of enrichment of IGF2BP binding sites and m 6 A modifications across mRNA transcriptome. ( i ) Percentages of various RNA species bound by IGF2BPs. ( j ) The distribution (upper) and enrichment (lower) of IGF2BPs binding peaks within different gene reions. The enrichment was determined by the proportion of IGF2BPs binding peaks normalized by the length of the region. Analyses in i and j were performed twice with similar results. ( k ) In vivo binding of Flag-IGF2BP2 to representative target genes in METTL14 knockdown or control HEK293T cells. Values are mean±s.d. of n =3 independent experiments. *, P

    Article Snippet: PolyA RNA was subsequently purified from 50–100 ng total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module.

    Techniques: Binding Assay, Modification, Affinity Chromatography, Methylation, Silver Staining, Western Blot, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Cross-linking Immunoprecipitation, In Vivo

    Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Journal: eLife

    Article Title: FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory

    doi: 10.7554/eLife.46919

    Figure Lengend Snippet: Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

    Article Snippet: Sequencing libraries were prepared using Poly(A) RNA selection (Dynabeads mRNA Purification Kit, ThermoFisher Scientific) and the TruSeq RNA Library Prep Kit (Illumina).

    Techniques: Immunoprecipitation, Titration, Incubation, Protein Extraction, Isolation, Protein Concentration, Western Blot, Concentration Assay

    Validated miRNA targets and pathways overlap with sporadic YOAD transcriptome changes. a Five-set Venn diagram showing 2899 mRNAs identified as differentially expressed in the posterior cingulate cortex (PCC) overlap of published microarray data (GSE39420). A total of 874 miRNA targets altered in the exosomes of CSF from sporadic YOAD patients overlapped with the entire microarray dataset. b KEGG pathways were identified using DAVID (v6.8) with the 874 overlapping validated miRNA targets altered in CSF-derived exosomes from sporadic YOAD. Enriched KEGG pathways ( y -axis) represented as −log 10 ( p value) ( x -axis). The number of genes shared for each pathway is shown at the end of each pathway bar

    Journal: Molecular Neurobiology

    Article Title: MicroRNA Expression Levels Are Altered in the Cerebrospinal Fluid of Patients with Young-Onset Alzheimer’s Disease

    doi: 10.1007/s12035-018-1032-x

    Figure Lengend Snippet: Validated miRNA targets and pathways overlap with sporadic YOAD transcriptome changes. a Five-set Venn diagram showing 2899 mRNAs identified as differentially expressed in the posterior cingulate cortex (PCC) overlap of published microarray data (GSE39420). A total of 874 miRNA targets altered in the exosomes of CSF from sporadic YOAD patients overlapped with the entire microarray dataset. b KEGG pathways were identified using DAVID (v6.8) with the 874 overlapping validated miRNA targets altered in CSF-derived exosomes from sporadic YOAD. Enriched KEGG pathways ( y -axis) represented as −log 10 ( p value) ( x -axis). The number of genes shared for each pathway is shown at the end of each pathway bar

    Article Snippet: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

    Techniques: Periodic Counter-current Chromatography, Microarray, Derivative Assay

    gas6 may only have subtle roles in caudal hindbrain development. a WT and gas6 mutant embryos were assayed for expression of valentino (r5/r6) (i), hoxb3a (r5-spinal cord) (ii), hoxa3 (r5/r6) (iii), islet1 (cranial nerves)(iv) and dm20 (oligodendrocyte marker) (vi) by ISH, as well as for the presence of OPCs and abducens neurons by crossing to the Tg (olig2:EGFP) vu12 line (v). In column (iv), yellow brackets mark cranial nerve V, blue brackets mark cranial nerve VII and red brackets mark cranial nerve X. White brackets indicate the presence of abducens (cranial nerve VI) in column (v). b Schemes showing RNA-seq library synthesis. Hindbrain tissue was dissected from 48 hpf gas6 mutant embryos in the olig2:eGFP background. Total RNA was collected from pools of hindbrain tissue and was used in library synthesis following the TruSeq Stranded mRNA Library Prep Kit (Illumina) protocol. c 1590 differentially expressed genes were identified from RNA-Seq where 41 out of the 928 up-regulated genes and 78 out of the 662 down-regulated genes were expressed in the hindbrain. GO terms related to Biological Processes were identified in both up-regulated and down-regulated genes using DAVID. d A subset of differentially expressed genes was validated via qPCR from independently collected hindbrain tissue samples. e ISH analysis of representative differentially expressed hindbrain genes (i) neurod6b , (ii) atoh1b and (iii) olig4 show no detectable change in expression pattern in gas6 mutant embryos

    Journal: Neural Development

    Article Title: Analysis of novel caudal hindbrain genes reveals different regulatory logic for gene expression in rhombomere 4 versus 5/6 in embryonic zebrafish

    doi: 10.1186/s13064-018-0112-y

    Figure Lengend Snippet: gas6 may only have subtle roles in caudal hindbrain development. a WT and gas6 mutant embryos were assayed for expression of valentino (r5/r6) (i), hoxb3a (r5-spinal cord) (ii), hoxa3 (r5/r6) (iii), islet1 (cranial nerves)(iv) and dm20 (oligodendrocyte marker) (vi) by ISH, as well as for the presence of OPCs and abducens neurons by crossing to the Tg (olig2:EGFP) vu12 line (v). In column (iv), yellow brackets mark cranial nerve V, blue brackets mark cranial nerve VII and red brackets mark cranial nerve X. White brackets indicate the presence of abducens (cranial nerve VI) in column (v). b Schemes showing RNA-seq library synthesis. Hindbrain tissue was dissected from 48 hpf gas6 mutant embryos in the olig2:eGFP background. Total RNA was collected from pools of hindbrain tissue and was used in library synthesis following the TruSeq Stranded mRNA Library Prep Kit (Illumina) protocol. c 1590 differentially expressed genes were identified from RNA-Seq where 41 out of the 928 up-regulated genes and 78 out of the 662 down-regulated genes were expressed in the hindbrain. GO terms related to Biological Processes were identified in both up-regulated and down-regulated genes using DAVID. d A subset of differentially expressed genes was validated via qPCR from independently collected hindbrain tissue samples. e ISH analysis of representative differentially expressed hindbrain genes (i) neurod6b , (ii) atoh1b and (iii) olig4 show no detectable change in expression pattern in gas6 mutant embryos

    Article Snippet: For each RNA-seq experiment, three libraries were synthesized from 3μg RNA for each WT and mutant sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina).

    Techniques: Mutagenesis, Expressing, Marker, In Situ Hybridization, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    RNA-seq profiling of individual kdm2aa -deficient embryos and their siblings. (A) Summary of experimental design. TruSeq stranded mRNA sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b , with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb . 2 , a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.

    Journal: PLoS Genetics

    Article Title: Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish

    doi: 10.1371/journal.pgen.1006959

    Figure Lengend Snippet: RNA-seq profiling of individual kdm2aa -deficient embryos and their siblings. (A) Summary of experimental design. TruSeq stranded mRNA sequencing libraries were prepared from four individual embryos from each genotype at both 5 d.p.f. and 12 d.p.f., totalling 48 libraries. Paired end sequencing with a read length of 75 bp was performed on four lanes of Illumina HiSeq 2500 machines. Sequence was aligned to the GRCz10 reference genome with TopHat and read counts were obtained with HTseq-count. Quality control was performed using QoRTs and outliers removed. 32,261 genes were detected. DESeq2 was used to determine DE genes in each individual clutch, DE genes at each stage by combining the two clutches for each stage whilst controlling for clutch in the DESeq2 model, and also in a combined analysis combining all 4 clutches whilst controlling for stage and clutch. (B) Venn diagram showing the overlap in DE genes between the two 5 d.p.f. experiments and the combined 5 d.p.f. analysis. The increase in power due to the increased sample size enabled an additional 1106 DE genes to be detected. (C) Venn diagram displaying the overlap in DE genes between the two 12 d.p.f. experiments and the combined 12 d.p.f. analysis. An additional 1102 DE genes were detected in the combined analysis. (D) Volcano plot of all detected genes in the combined RNA-seq analysis with the 3752 DE genes shown in red and kdm2aa circled. (E) Box plot of normalised counts for the chromatin modifier nsd1b , with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (F) Box plot of normalised counts for dnmt3bb . 2 , a de novo DNA methyl transferase, with adjusted p-values for individual experiments, stage-specific and combined analysis as indicated by horizontal bars. Data for heterozygous and wild-type siblings are combined. In the figure legend ‘s’ denotes siblings and ‘m’ homozygous mutants. (G) Table of enriched Gene Ontology (GO) terms of the biological process (BP) GO domain which overlap in both 5 d.p.f. experiments and also the 5 d.p.f. combined analysis. P-values shown are for the combined analysis. (H) Table of enriched GO terms that overlap in both individual 12 d.p.f. experiments and the 12 d.p.f. combined analysis, with p-values for the combined analysis shown.

    Article Snippet: From these 48 samples 300 ng total RNA were used to prepare sequencing libraries with Ambion ERCC spike-in mix 1 (Cat. No. 4456740) according to the manufacturer’s instructions using the Illumina TruSeq Stranded mRNA Sample Prep Kit Set A and B (RS-122-2101 and RS-122-2102).

    Techniques: RNA Sequencing Assay, Sequencing

    Mutations in SNU13 inhibit pre-mRNA splicing. ( A ) Primer extension analysis of pre-U3 snoRNA splicing. Strain YGALSNU13 was grown in the presence of plasmid-based wild-type SNU13 or mutants E59A, R84A/V81L, or Δ111-126. Total RNA was harvested

    Journal: RNA

    Article Title: Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

    doi: 10.1261/rna.5970404

    Figure Lengend Snippet: Mutations in SNU13 inhibit pre-mRNA splicing. ( A ) Primer extension analysis of pre-U3 snoRNA splicing. Strain YGALSNU13 was grown in the presence of plasmid-based wild-type SNU13 or mutants E59A, R84A/V81L, or Δ111-126. Total RNA was harvested

    Article Snippet: Poly(A)+ mRNA was prepared from 200 μg total RNA by using the GenElute mRNA miniprep kit (Sigma) for YGALSNU13 grown for 12 h at 30°C in the presence of plasmid based SNU13 mutant E59A or wild-type SNU13 in YPD or YPGal, respectively.

    Techniques: Plasmid Preparation

    Distinct nucleotide composition patterns at mRNA 3′-end regions. Nucleotide composition plot of poly(A) site, −50 to +50 window for Direct RNA sequencing data from human liver RNA sample in A ) and 3Seq data from human U2OS cell line in B ). ( C ) 3Seq metagene analysis of −150 to +100 window centered at the cleavage site. The percent reads are calculated by dividing the read coverage on each position by all the number of reads mapped to this window. 3Seq reads are significantly enriched in the −100 to 0 position, with dramatic drop downstream from the 0 position. ( D ) Percent reads plot of 3′ ends of trimmed reads centered at the cleavage site whose +1 position is T, G, or C. ( E ) H3K36me3 metagene plot centered at the cleavage site. Distal peaks (blue) are defined as the most 3′ peak assigned to a given gene. Proximal peaks (red) are peaks mapped to RefSeq and Ensembl-annotated that are proximal in location to the distal peaks.

    Journal: RNA

    Article Title: Genome-wide maps of polyadenylation reveal dynamic mRNA 3?-end formation in mammalian cell lineages

    doi: 10.1261/rna.035360.112

    Figure Lengend Snippet: Distinct nucleotide composition patterns at mRNA 3′-end regions. Nucleotide composition plot of poly(A) site, −50 to +50 window for Direct RNA sequencing data from human liver RNA sample in A ) and 3Seq data from human U2OS cell line in B ). ( C ) 3Seq metagene analysis of −150 to +100 window centered at the cleavage site. The percent reads are calculated by dividing the read coverage on each position by all the number of reads mapped to this window. 3Seq reads are significantly enriched in the −100 to 0 position, with dramatic drop downstream from the 0 position. ( D ) Percent reads plot of 3′ ends of trimmed reads centered at the cleavage site whose +1 position is T, G, or C. ( E ) H3K36me3 metagene plot centered at the cleavage site. Distal peaks (blue) are defined as the most 3′ peak assigned to a given gene. Proximal peaks (red) are peaks mapped to RefSeq and Ensembl-annotated that are proximal in location to the distal peaks.

    Article Snippet: For the 3Seq experiment, 0.5–2 μg of total RNA are poly(A) selected twice using the Invitrogen Dynabeads mRNA DIRECT Kit.

    Techniques: RNA Sequencing Assay

    Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Expressing

    a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Incubation, Fluorescence, Labeling, Expressing, Transfection

    Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Positive Control, Negative Control, Confocal Laser Scanning Microscopy, Fluorescence, Staining

    Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Incubation, Transfection, Flow Cytometry, Cytometry

    The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Positive Control, Negative Control, Fluorescence, Expressing

    Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Transfection

    Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transmission Electron Microscopy, Staining

    Effects of intrathecal ATL on the expression of IL-1β, IL-6 and TNF-α mRNA assessed by RT-PCR in the spinal cords of rats with CIBP. The L4-L5 spinal cords were used to test 2 hours after i.t. injection with ATL (0.3 nmol/20 μl) or NS (20 μl) on day 11 after surgery. Data are expressed as means ± SEM. ** P

    Journal: Journal of Neuroinflammation

    Article Title: Lipoxins and aspirin-triggered lipoxin alleviate bone cancer pain in association with suppressing expression of spinal proinflammatory cytokines

    doi: 10.1186/1742-2094-9-278

    Figure Lengend Snippet: Effects of intrathecal ATL on the expression of IL-1β, IL-6 and TNF-α mRNA assessed by RT-PCR in the spinal cords of rats with CIBP. The L4-L5 spinal cords were used to test 2 hours after i.t. injection with ATL (0.3 nmol/20 μl) or NS (20 μl) on day 11 after surgery. Data are expressed as means ± SEM. ** P

    Article Snippet: Quantification of mRNA levels of IL-1β, IL-6, TNF-α and GAPDH were analyzed by SYBR Green qRT-PCR detection (iCycler iQ® real-time PCR detection system, Bio-Rad, Hercules City, California, USA), with each sample being run in duplicate.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection

    Effects of ROCK1 and ZIPK knockdown on IL-6 and IL-1β mRNA expression and IL-6 protein secretion. (A) CASMCs were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA. Cells were lysed 48 h later for qRT-PCR to quantify IL-6 and IL-1β mRNA levels. Values represent means ± SEM ( n = 7 for IL-6 and n = 6 for IL-1β). *significantly different from control (p

    Journal: PLoS ONE

    Article Title: The Effects of Knockdown of Rho-Associated Kinase 1 and Zipper-Interacting Protein Kinase on Gene Expression and Function in Cultured Human Arterial Smooth Muscle Cells

    doi: 10.1371/journal.pone.0116969

    Figure Lengend Snippet: Effects of ROCK1 and ZIPK knockdown on IL-6 and IL-1β mRNA expression and IL-6 protein secretion. (A) CASMCs were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA. Cells were lysed 48 h later for qRT-PCR to quantify IL-6 and IL-1β mRNA levels. Values represent means ± SEM ( n = 7 for IL-6 and n = 6 for IL-1β). *significantly different from control (p

    Article Snippet: Levels of mRNA expression were quantified by RT-PCR using a BioRad iCycler MyiQ detection system (BioRad) with pre-synthesized primers (Applied Biosystems) for ZIPK (Hs00154676_m1), IL-1β (Hs00174097_m1) and IL-6 (Hs00174131_m1).

    Techniques: Expressing, Transfection, Negative Control, Quantitative RT-PCR

    The novel RNA species is devoid of a poly-A tail dstet5 cells were treated with 10 μM LAM or 0.1 μM ETV for 3 days in the absence of tet. Total cellular RNA were extracted with TRIzol reagents and polyA + mRNA were extracted with an Oligotex mRNA mini kit from total RNA preparations. Each of the lanes was loaded with 6 μg of total cellular RNA or polyA-selected RNA from 6 μg of total cellular RNA from dstet5 cells left untreated or treated with the indicated concentrations of viral DNA polymerase inhibitors. To ensure each of the lanes are loaded with equal amount of RNA, each of the polyA-selected RNA samples was mixed with 6 μg of total cellular RNA prepared from parental LMH cells. DHBV RNA were detected by Northern blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.

    Journal: Antiviral research

    Article Title: Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors

    doi: 10.1016/j.antiviral.2016.04.007

    Figure Lengend Snippet: The novel RNA species is devoid of a poly-A tail dstet5 cells were treated with 10 μM LAM or 0.1 μM ETV for 3 days in the absence of tet. Total cellular RNA were extracted with TRIzol reagents and polyA + mRNA were extracted with an Oligotex mRNA mini kit from total RNA preparations. Each of the lanes was loaded with 6 μg of total cellular RNA or polyA-selected RNA from 6 μg of total cellular RNA from dstet5 cells left untreated or treated with the indicated concentrations of viral DNA polymerase inhibitors. To ensure each of the lanes are loaded with equal amount of RNA, each of the polyA-selected RNA samples was mixed with 6 μg of total cellular RNA prepared from parental LMH cells. DHBV RNA were detected by Northern blot hybridization. Ribosomal RNAs (28S and 18S rRNA) served as loading controls. The positions of DHBV pgRNA and subgenomic mRNA encoding viral envelope proteins (envRNA) are indicated. The RNA species accumulated in the cells treated with DNA polymerase inhibitors are indicated as novel RNA.

    Article Snippet: To determine whether these novel RNA species contain a poly A+ tail, poly A+ mRNA was extracted from total RNA preparations with Oligotex mRNA Kit (Qiagen).

    Techniques: Laser Capture Microdissection, Northern Blot, Hybridization

    Experimental design and construction of the pAs reference Study design. 3′ READS method was used to identify the pAs expressed in the fibroblasts from C57BL/6J and SPRET/EiJ mouse strains. 3′ mRNA‐Seq method was used to quantify the usage of the identified pAs in both two parental strains and their F1 hybrids. The frequencies of 13 known PAS motifs in the 100 nt upstream region of pAs identified in C57BL/6J. pseudo_pAs represents pAs identified by using reads without non‐genomic T ( Materials and Methods ). raw_pAs and filtered_pAs represent the pAs determined by the PASS reads with and without further filtering, respectively ( Materials and Methods ). X ‐axis shows different types of PAS motifs, and y ‐axis shows the percentage of pAs with the specific motif in the upstream region. See Fig EV1 C for the results from SPRET/EiJ. 13,406 pAs identified in this study were located within 50 nt away from the 3′ end of RNA transcripts annotated in Ensembl. X ‐axis shows the distance between the 13,406 pAs identified in this study and the annotated pAs. Y ‐axis represents the density. Inset: Barplot shows the number of pAs with representative cleavage site identical to the annotated transcript ends as well as those within 5 nt upstream or downstream to the annotated ends, respectively. The pie chart shows the percentage of protein‐coding genes with different number of pAs. Schematic definition for different types of pAs. See Materials and Methods for the details. Pie chart shows the percentage of different types of pAs identified in this study.

    Journal: Molecular Systems Biology

    Article Title: Global analysis of regulatory divergence in the evolution of mouse alternative polyadenylation

    doi: 10.15252/msb.20167375

    Figure Lengend Snippet: Experimental design and construction of the pAs reference Study design. 3′ READS method was used to identify the pAs expressed in the fibroblasts from C57BL/6J and SPRET/EiJ mouse strains. 3′ mRNA‐Seq method was used to quantify the usage of the identified pAs in both two parental strains and their F1 hybrids. The frequencies of 13 known PAS motifs in the 100 nt upstream region of pAs identified in C57BL/6J. pseudo_pAs represents pAs identified by using reads without non‐genomic T ( Materials and Methods ). raw_pAs and filtered_pAs represent the pAs determined by the PASS reads with and without further filtering, respectively ( Materials and Methods ). X ‐axis shows different types of PAS motifs, and y ‐axis shows the percentage of pAs with the specific motif in the upstream region. See Fig EV1 C for the results from SPRET/EiJ. 13,406 pAs identified in this study were located within 50 nt away from the 3′ end of RNA transcripts annotated in Ensembl. X ‐axis shows the distance between the 13,406 pAs identified in this study and the annotated pAs. Y ‐axis represents the density. Inset: Barplot shows the number of pAs with representative cleavage site identical to the annotated transcript ends as well as those within 5 nt upstream or downstream to the annotated ends, respectively. The pie chart shows the percentage of protein‐coding genes with different number of pAs. Schematic definition for different types of pAs. See Materials and Methods for the details. Pie chart shows the percentage of different types of pAs identified in this study.

    Article Snippet: In order to reduce such potential errors, we created a mock F1 hybrid dataset by pooling the 3′ mRNA‐Seq reads from the two parental strains together and then performed the same mapping procedure as that for the real F1 hybrid dataset.

    Techniques:

    Dissection of cis‐ and trans ‐contribution in APA divergence Two representative examples of parental divergent pAs events in gene Txndc16 (up) and Gatb (down). In the left panel, above each gene structure, the red triangles represent the identified pAs clusters and the black bars represent 3′ mRNA‐Seq reads mapped within distinct pAs clusters. Pink and blue shades mark proximal and distal pAs, respectively. The right panel shows the independent validation of the two divergent pAs using 3′ RACE method. The gel images illustrate 3′ RACE products obtained from C57BL/6J (BL) and SPRET/EiJ (SP) fibroblasts. Positions of the distal and the proximal pAs isoforms are indicated with blue and pink arrows, respectively. Scatterplot showing the comparison of allelic difference (log2 C57BL/6J (BL) and SPRET/EiJ (SP)) in distal/proximal pAs usage ratio estimated by 3′ mRNA‐Seq ( x ‐axis) and that by PacBio sequencing ( y ‐axis). Each circle represents one gene, and in total, 20 candidate genes were randomly chosen for validation. Scatterplot comparing the parental difference in pAs usage to the allelic difference in F1 hybrids ( x ‐axis). Out of 2,532 divergent APA events between the parental strains, 1,876 (indicated as “+”) and 572 (indicated as “x”) exhibit significant cis ‐ and trans ‐regulatory divergence, respectively. Percentage of the five types of pAs events regulated by cis ‐ and trans ‐divergence (numbers of events for each type are labeled above the bars).

    Journal: Molecular Systems Biology

    Article Title: Global analysis of regulatory divergence in the evolution of mouse alternative polyadenylation

    doi: 10.15252/msb.20167375

    Figure Lengend Snippet: Dissection of cis‐ and trans ‐contribution in APA divergence Two representative examples of parental divergent pAs events in gene Txndc16 (up) and Gatb (down). In the left panel, above each gene structure, the red triangles represent the identified pAs clusters and the black bars represent 3′ mRNA‐Seq reads mapped within distinct pAs clusters. Pink and blue shades mark proximal and distal pAs, respectively. The right panel shows the independent validation of the two divergent pAs using 3′ RACE method. The gel images illustrate 3′ RACE products obtained from C57BL/6J (BL) and SPRET/EiJ (SP) fibroblasts. Positions of the distal and the proximal pAs isoforms are indicated with blue and pink arrows, respectively. Scatterplot showing the comparison of allelic difference (log2 C57BL/6J (BL) and SPRET/EiJ (SP)) in distal/proximal pAs usage ratio estimated by 3′ mRNA‐Seq ( x ‐axis) and that by PacBio sequencing ( y ‐axis). Each circle represents one gene, and in total, 20 candidate genes were randomly chosen for validation. Scatterplot comparing the parental difference in pAs usage to the allelic difference in F1 hybrids ( x ‐axis). Out of 2,532 divergent APA events between the parental strains, 1,876 (indicated as “+”) and 572 (indicated as “x”) exhibit significant cis ‐ and trans ‐regulatory divergence, respectively. Percentage of the five types of pAs events regulated by cis ‐ and trans ‐divergence (numbers of events for each type are labeled above the bars).

    Article Snippet: In order to reduce such potential errors, we created a mock F1 hybrid dataset by pooling the 3′ mRNA‐Seq reads from the two parental strains together and then performed the same mapping procedure as that for the real F1 hybrid dataset.

    Techniques: Dissection, Sequencing, Labeling

    Global analysis of RNA-seq and ribosome profiling data a High reproducibility of the ribo-seq experiment under hypoxic stress: Pearson correlation coefficient is shown between two independent ribo-seq experiments under hypoxic stress for 1 h. b Length distribution of mRNA and RPF reads. Analysis of the insert size distribution of RPF reads and mRNA reads across all replicates and time points. The majority of RPF reads are 28 ~ 32 nt in length. c Reads distribution and ( d ) normalized read density of RPF reads and mRNA reads on genomic regions of 5’UTRs, CDSs, 3′ UTRs and introns of mRNAs are shown. RPF reads are presented on the left, and mRNA reads are presented on the right

    Journal: BMC Genomics

    Article Title: Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress

    doi: 10.1186/s12864-017-3996-8

    Figure Lengend Snippet: Global analysis of RNA-seq and ribosome profiling data a High reproducibility of the ribo-seq experiment under hypoxic stress: Pearson correlation coefficient is shown between two independent ribo-seq experiments under hypoxic stress for 1 h. b Length distribution of mRNA and RPF reads. Analysis of the insert size distribution of RPF reads and mRNA reads across all replicates and time points. The majority of RPF reads are 28 ~ 32 nt in length. c Reads distribution and ( d ) normalized read density of RPF reads and mRNA reads on genomic regions of 5’UTRs, CDSs, 3′ UTRs and introns of mRNAs are shown. RPF reads are presented on the left, and mRNA reads are presented on the right

    Article Snippet: RPF and mRNA libraries were sequenced on an Illumina HiSeq 2500 sequencer with TruSeq SBS Kit v3 for single-end 50 cycles (SE50) run type.

    Techniques: RNA Sequencing Assay

    Correlation between transcriptional and translational changes. Differentially expressed genes (DEGs) under 1 h, 2 h and 4 h hypoxic stress at mRNA level a RPF level b and DTE level c Up panel: Venn diagram of overlapped genes across three time points; bottom panel: heatmap of DEGs presented at three time points. d Correlations between mRNA and RPF level changes. Pearson’s correlation were compared by gene set presented in the Venn diagrams. Statistical analysis was performed using Wilcox test: + means p

    Journal: BMC Genomics

    Article Title: Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress

    doi: 10.1186/s12864-017-3996-8

    Figure Lengend Snippet: Correlation between transcriptional and translational changes. Differentially expressed genes (DEGs) under 1 h, 2 h and 4 h hypoxic stress at mRNA level a RPF level b and DTE level c Up panel: Venn diagram of overlapped genes across three time points; bottom panel: heatmap of DEGs presented at three time points. d Correlations between mRNA and RPF level changes. Pearson’s correlation were compared by gene set presented in the Venn diagrams. Statistical analysis was performed using Wilcox test: + means p

    Article Snippet: RPF and mRNA libraries were sequenced on an Illumina HiSeq 2500 sequencer with TruSeq SBS Kit v3 for single-end 50 cycles (SE50) run type.

    Techniques:

    microHIVE differentiation of spinal motor neurons. (A) Target and experimental molecular profiles of retinoic acid and GDF11. (Left) Based on correlative analysis of growth factor concentrations and RNA profiling, we designed a continuous profile of retinoic acid and GDF11 to induce rostral-caudal patterning of motor neurons, to correspond to the brachial, thoracic and lumbar regions of the spinal cord. (Right) The microHIVE experimental profile showed a close correlation to the desired target profile (profile similarity metric, ε = 0.991). (B) Schematic illustration of chamber binning. After motor neuron differentiation (day 28), the all-polymer cell culture chamber could be sectioned into seven bins for cellular characterization and spatial correlation. (C) Differential expressions of HOX genes across the chamber bins. HOX gene expressions were profiled from the respective chamber sections through quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH mRNA levels, and subsequently gene (column) normalized across all bins to compare respective gene expression trends in the form of a heat map. (D) Immunofluorescence confirmation of HOX protein expressions. Immunostaining of HOXB4 and HOXC8 (red) and SMI-32 (green) across the seven culture bins demonstrated successful generation of rostral (HOXB4) to caudal (HOXC8) motor neurons. Cellular nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Cellular quantification based on positive staining of HOXB4 and HOXC8 further confirmed the respective protein expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in A and D .

    Journal: Theranostics

    Article Title: Microhexagon gradient array directs spatial diversification of spinal motor neurons

    doi: 10.7150/thno.29755

    Figure Lengend Snippet: microHIVE differentiation of spinal motor neurons. (A) Target and experimental molecular profiles of retinoic acid and GDF11. (Left) Based on correlative analysis of growth factor concentrations and RNA profiling, we designed a continuous profile of retinoic acid and GDF11 to induce rostral-caudal patterning of motor neurons, to correspond to the brachial, thoracic and lumbar regions of the spinal cord. (Right) The microHIVE experimental profile showed a close correlation to the desired target profile (profile similarity metric, ε = 0.991). (B) Schematic illustration of chamber binning. After motor neuron differentiation (day 28), the all-polymer cell culture chamber could be sectioned into seven bins for cellular characterization and spatial correlation. (C) Differential expressions of HOX genes across the chamber bins. HOX gene expressions were profiled from the respective chamber sections through quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH mRNA levels, and subsequently gene (column) normalized across all bins to compare respective gene expression trends in the form of a heat map. (D) Immunofluorescence confirmation of HOX protein expressions. Immunostaining of HOXB4 and HOXC8 (red) and SMI-32 (green) across the seven culture bins demonstrated successful generation of rostral (HOXB4) to caudal (HOXC8) motor neurons. Cellular nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Cellular quantification based on positive staining of HOXB4 and HOXC8 further confirmed the respective protein expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in A and D .

    Article Snippet: For bin-based RNA sequencing, polyA enrichment of mRNA was performed for Illumina sequencing library preparation, according to the manufacturer's kit instructions with standard index sequences (Illumina).

    Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Immunostaining, Staining

    Derivation of spinal motor neurons from human pluripotent stem cells. (A) Schematic illustration of spinal motor neuron differentiation from induced pluripotent stem cell (iPSC). Varying concentrations of retinoic acid and GDF11 were used to generate thoracic/lumbar motor neurons. (B) Immunofluorescence analysis of cellular identities. (Top) Co-staining of Nestin (green) and SOX1 (red) confirmed the generation of neural progenitor cells (NPC) at day 10 of cellular differentiation. (Bottom) Co-staining of SMI-32 (green) and ISL1 (red) at day 28 demonstrated successful differentiation of iPSCs into spinal motor neurons (MN). Cellular nuclei were counterstained with DAPI (blue). All scale bars indicate 50 μm. (C) RNA confirmation of cellular identities. mRNA expression levels of the corresponding neural progenitor and motor neuron markers were measured by quantitative PCR. All gene expression analyses were normalized to that of iPSC mRNA levels. (**** P

    Journal: Theranostics

    Article Title: Microhexagon gradient array directs spatial diversification of spinal motor neurons

    doi: 10.7150/thno.29755

    Figure Lengend Snippet: Derivation of spinal motor neurons from human pluripotent stem cells. (A) Schematic illustration of spinal motor neuron differentiation from induced pluripotent stem cell (iPSC). Varying concentrations of retinoic acid and GDF11 were used to generate thoracic/lumbar motor neurons. (B) Immunofluorescence analysis of cellular identities. (Top) Co-staining of Nestin (green) and SOX1 (red) confirmed the generation of neural progenitor cells (NPC) at day 10 of cellular differentiation. (Bottom) Co-staining of SMI-32 (green) and ISL1 (red) at day 28 demonstrated successful differentiation of iPSCs into spinal motor neurons (MN). Cellular nuclei were counterstained with DAPI (blue). All scale bars indicate 50 μm. (C) RNA confirmation of cellular identities. mRNA expression levels of the corresponding neural progenitor and motor neuron markers were measured by quantitative PCR. All gene expression analyses were normalized to that of iPSC mRNA levels. (**** P

    Article Snippet: For bin-based RNA sequencing, polyA enrichment of mRNA was performed for Illumina sequencing library preparation, according to the manufacturer's kit instructions with standard index sequences (Illumina).

    Techniques: Immunofluorescence, Staining, Cell Differentiation, Expressing, Real-time Polymerase Chain Reaction

    u-Eleanor plays a role in coordinated up-regulation of intragenic Eleanor and ESR1 mRNA to promote the proliferative activity of LTED cells. ( a ) Overview of a region upstream of the ESR1 locus. The RNA-Seq tracks were aligned with the ChIP-Seq data available in the UCSC genome browser 39 (University of California, Santa Cruz, CA, and Supplementary Table 1 ). Sites amplified by qPCR are shown ( a – f ). ( b ) Local expression of u-Eleanor . u-Eleanor was induced at site c in LTED cells, repressed in LTED-RES cells, and de-repressed by ICI 182,780 treatment (ER antagonist, related to Fig. 5e–g ). For qRT–PCR, total RNA was pre-treated with DNase I, and the amplification efficiency for each primer set was normalized. The value for site b in MCF7 was set to 1. Values are the means±s.d.; n =3. Corresponding ΔC t values are listed in Supplementary Table 6 . ( c ) RNA Pol II binding to the u-Eleanor region ( c ) and ESR1 promoter A (f). For ChIP-qPCR, values are the means±s.d.; n =3. P -values were calculated using Student's t -test (** P

    Journal: Nature Communications

    Article Title: A cluster of noncoding RNAs activates the ESR1 locus during breast cancer adaptation

    doi: 10.1038/ncomms7966

    Figure Lengend Snippet: u-Eleanor plays a role in coordinated up-regulation of intragenic Eleanor and ESR1 mRNA to promote the proliferative activity of LTED cells. ( a ) Overview of a region upstream of the ESR1 locus. The RNA-Seq tracks were aligned with the ChIP-Seq data available in the UCSC genome browser 39 (University of California, Santa Cruz, CA, and Supplementary Table 1 ). Sites amplified by qPCR are shown ( a – f ). ( b ) Local expression of u-Eleanor . u-Eleanor was induced at site c in LTED cells, repressed in LTED-RES cells, and de-repressed by ICI 182,780 treatment (ER antagonist, related to Fig. 5e–g ). For qRT–PCR, total RNA was pre-treated with DNase I, and the amplification efficiency for each primer set was normalized. The value for site b in MCF7 was set to 1. Values are the means±s.d.; n =3. Corresponding ΔC t values are listed in Supplementary Table 6 . ( c ) RNA Pol II binding to the u-Eleanor region ( c ) and ESR1 promoter A (f). For ChIP-qPCR, values are the means±s.d.; n =3. P -values were calculated using Student's t -test (** P

    Article Snippet: Preparation of mRNA-Seq libraries Total RNA was extracted from cultured cells with an RNeasy Mini Kit (Qiagen). mRNA-Seq libraries were generated using an mRNA-Seq Sample Preparation Kit (Illumina) according to manufacturer's protocol with minor modifications.

    Techniques: Activity Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Binding Assay

    Pathway-based gene set enrichment analyses of differentially expressed mRNAs or miRNAs. Protein-coding genes were ranked according to the number of pathways in which they were prioritized as core genes (most significantly deregulated genes). miRNAs were ranked according to the number of pathways in which their target genes were prioritized as core genes. Columns in green/red represent the genes or miRNAs that were down/upregulated on average in at least two ccRCCs respectively; columns in black represent the genes or miRNA targets involved in at least one cancer-associated pathway. The height of the columns in different colors represents the number of pathways where the genes or miRNA targets were ranked as core genes. A: Genes ranked in the top 20 based on the results of pathway-based gene set enrichment analysis. B: miRNAs ranked the top 20 based on the results of pathway-based gene set enrichment analysis of their target genes.

    Journal: PLoS ONE

    Article Title: Integrated Profiling of MicroRNAs and mRNAs: MicroRNAs Located on Xq27.3 Associate with Clear Cell Renal Cell Carcinoma

    doi: 10.1371/journal.pone.0015224

    Figure Lengend Snippet: Pathway-based gene set enrichment analyses of differentially expressed mRNAs or miRNAs. Protein-coding genes were ranked according to the number of pathways in which they were prioritized as core genes (most significantly deregulated genes). miRNAs were ranked according to the number of pathways in which their target genes were prioritized as core genes. Columns in green/red represent the genes or miRNAs that were down/upregulated on average in at least two ccRCCs respectively; columns in black represent the genes or miRNA targets involved in at least one cancer-associated pathway. The height of the columns in different colors represents the number of pathways where the genes or miRNA targets were ranked as core genes. A: Genes ranked in the top 20 based on the results of pathway-based gene set enrichment analysis. B: miRNAs ranked the top 20 based on the results of pathway-based gene set enrichment analysis of their target genes.

    Article Snippet: In this report, we present an integrative analysis of digital gene expression (DGE) profiling of both mRNAs and miRNAs in ccRCC by initially detecting their expression levels in 10 matched tumor-normal (adjacent) tissue pairs using the Illumina GA II platform and then validating some of our most interesting findings in a large cohort of ccRCC patients.

    Techniques:

    Overview of the expression profiles of miRNAs and mRNAs in 10 ccRCC patients. A: The number of miRNAs and mRNAs differentially expressed in 10 ccRCC patients ( P

    Journal: PLoS ONE

    Article Title: Integrated Profiling of MicroRNAs and mRNAs: MicroRNAs Located on Xq27.3 Associate with Clear Cell Renal Cell Carcinoma

    doi: 10.1371/journal.pone.0015224

    Figure Lengend Snippet: Overview of the expression profiles of miRNAs and mRNAs in 10 ccRCC patients. A: The number of miRNAs and mRNAs differentially expressed in 10 ccRCC patients ( P

    Article Snippet: In this report, we present an integrative analysis of digital gene expression (DGE) profiling of both mRNAs and miRNAs in ccRCC by initially detecting their expression levels in 10 matched tumor-normal (adjacent) tissue pairs using the Illumina GA II platform and then validating some of our most interesting findings in a large cohort of ccRCC patients.

    Techniques: Expressing

    Comparison of deep sequencing data and qPCR results. For the comparison of deep sequencing data and qPCR results, genes determined to be differentially expressed in all of the 10 patients by deep sequencing were validated using qPCR. The height of the columns in the chart represents the log-transformed average fold change (tumor/normal) in expression across the 10 patients for each of the genes validated; bars represent standard errors. A: The validation results of six miRNAs indicated that the deep sequencing data were in excellent agreement with the qPCR results. B: The validation results of six mRNAs also indicated that the results of the deep sequencing were generally agreed well with the qPCR results.

    Journal: PLoS ONE

    Article Title: Integrated Profiling of MicroRNAs and mRNAs: MicroRNAs Located on Xq27.3 Associate with Clear Cell Renal Cell Carcinoma

    doi: 10.1371/journal.pone.0015224

    Figure Lengend Snippet: Comparison of deep sequencing data and qPCR results. For the comparison of deep sequencing data and qPCR results, genes determined to be differentially expressed in all of the 10 patients by deep sequencing were validated using qPCR. The height of the columns in the chart represents the log-transformed average fold change (tumor/normal) in expression across the 10 patients for each of the genes validated; bars represent standard errors. A: The validation results of six miRNAs indicated that the deep sequencing data were in excellent agreement with the qPCR results. B: The validation results of six mRNAs also indicated that the results of the deep sequencing were generally agreed well with the qPCR results.

    Article Snippet: In this report, we present an integrative analysis of digital gene expression (DGE) profiling of both mRNAs and miRNAs in ccRCC by initially detecting their expression levels in 10 matched tumor-normal (adjacent) tissue pairs using the Illumina GA II platform and then validating some of our most interesting findings in a large cohort of ccRCC patients.

    Techniques: Sequencing, Real-time Polymerase Chain Reaction, Transformation Assay, Expressing

    Expression levels of FSHβ (A, B) and LHβ (C, D)mRNAs in Nile tilapia pituitaries cultured with different concentrations of NKB (A, C) or NKBRP (B, D) peptides. Relative abundance of the mRNAs was normalized to the amount of GAPDH by the comparative threshold cycle method using qRTPCR. Results are means ± SEM (n=5–8). * indicates significant difference from 0 μM group (P

    Journal: Development & Reproduction

    Article Title: Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus

    doi: 10.12717/DR.2016.20.1.051

    Figure Lengend Snippet: Expression levels of FSHβ (A, B) and LHβ (C, D)mRNAs in Nile tilapia pituitaries cultured with different concentrations of NKB (A, C) or NKBRP (B, D) peptides. Relative abundance of the mRNAs was normalized to the amount of GAPDH by the comparative threshold cycle method using qRTPCR. Results are means ± SEM (n=5–8). * indicates significant difference from 0 μM group (P

    Article Snippet: The abundance level of the mRNAs was normalized against the amount of GAPDH and the relative abundance was determined by the comparative threshold cycle method, 2–△△Ct , using CFX Manager™ Software (Bio-Rad).

    Techniques: Expressing, Cell Culture

    Expression levels of GnRH I (A), Kiss2 (B) andtac3 (C) mRNAs in the brains and FSHβ (D) and LHβ (E) mRNAs in the pituitaries of Nile tilapia injected either with LHRH, NKB, NKBRP or PBS. Relative abundance of the mRNAs in the brains was normalized to the amount of GAPDH and β-actin and in the pituitaries was normalized to the amount of GAPDH by the comparative threshold cycle method using qRT-PCR. Results are means ± SEM (n=6–8). * indicates significant difference from PBS group (P

    Journal: Development & Reproduction

    Article Title: Neurokinin B-related Peptide Suppresses the Expression of GnRH I, Kiss2 and tac3 in the Brain of Mature Female Nile tilapia Oreochromis niloticus

    doi: 10.12717/DR.2016.20.1.051

    Figure Lengend Snippet: Expression levels of GnRH I (A), Kiss2 (B) andtac3 (C) mRNAs in the brains and FSHβ (D) and LHβ (E) mRNAs in the pituitaries of Nile tilapia injected either with LHRH, NKB, NKBRP or PBS. Relative abundance of the mRNAs in the brains was normalized to the amount of GAPDH and β-actin and in the pituitaries was normalized to the amount of GAPDH by the comparative threshold cycle method using qRT-PCR. Results are means ± SEM (n=6–8). * indicates significant difference from PBS group (P

    Article Snippet: The abundance level of the mRNAs was normalized against the amount of GAPDH and the relative abundance was determined by the comparative threshold cycle method, 2–△△Ct , using CFX Manager™ Software (Bio-Rad).

    Techniques: Expressing, Injection, Quantitative RT-PCR

    Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: Subtractive hybridization kits, such as the Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit, which until recently has been considered to be one of the best and most widely used choices, rely on oligonucleotide probes to capture 16S and 23S rRNA.

    Techniques: Isolation, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Article Snippet: Subtractive hybridization kits, such as the Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit, which until recently has been considered to be one of the best and most widely used choices, rely on oligonucleotide probes to capture 16S and 23S rRNA.

    Techniques: Isolation, Quantitation Assay, RNA HS Assay, Software, Fluorescence

    rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: Subtractive hybridization kits, such as the Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit, which until recently has been considered to be one of the best and most widely used choices, rely on oligonucleotide probes to capture 16S and 23S rRNA.

    Techniques: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

    The procedure for the isolation of antipodal cells, mRNA extraction and cDNA amplification. Briefly, the ovules are dissected from flowers at the stage 12c and are further dissected by handmade razor under fluorescent microscope to obtain ovule fragments containing antipodal cells. The antipodal cells are further separated from other tissues by microneedles. The isolated antipodal cells are transferred into the 2x lysis buffer. Dynabeads are used for mRNA extraction and SMARTer Ultra Low Input RNA kit for Sequencing-v3 is used for cDNA amplification subsequently.

    Journal: PLoS ONE

    Article Title: An Efficient Antipodal Cell Isolation Method for Screening of Cell Type-Specific Genes in Arabidopsis thaliana

    doi: 10.1371/journal.pone.0166390

    Figure Lengend Snippet: The procedure for the isolation of antipodal cells, mRNA extraction and cDNA amplification. Briefly, the ovules are dissected from flowers at the stage 12c and are further dissected by handmade razor under fluorescent microscope to obtain ovule fragments containing antipodal cells. The antipodal cells are further separated from other tissues by microneedles. The isolated antipodal cells are transferred into the 2x lysis buffer. Dynabeads are used for mRNA extraction and SMARTer Ultra Low Input RNA kit for Sequencing-v3 is used for cDNA amplification subsequently.

    Article Snippet: mRNA extraction and cDNA amplification mRNA of antipodal cells was extracted using Dynabeads mRNA DIRECT Micro Kit (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Isolation, Amplification, Microscopy, Lysis, Sequencing