first strand synthesis kit  (TaKaRa)

Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: AGO-CLASH identifies potential TDMD trigger RNAs. ( A ) Plots showing reads per million (RPM) of the sum of all chimeras detected for each miRNA in enriched versus unenriched AGO-CLASH brain and lung samples. Enriched miRNAs are shown in red. ( B ) Proportion of chimeras mapped to each location in replicate WT and Zswim8 − / − brain AGO-CLASH samples enriched with probe 1. Only chimeras in which the predicted base-pairing to the miRNA seed sequence was a 6mer, 7mer-A1, 7mer-m8, or 8mer were included in the analysis. ( C ) Table of filtered and ranked candidate TDMD trigger sites identified by AGO-CLASH. Shown are the top-ranked triggers for miR-7a-5p, miR-29b-3p, and miR-30b-5p, and the top three candidate triggers for miR-322-5p and miR-503-5p. ( D ) qRT-PCR analysis of candidate trigger sites in MEFs. Immortalized MEFs expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or two independent guides targeting candidate trigger RNAs (sg1 and sg2). Shown is the candidate trigger expression normalized to Actb ( left ), mature miRNA abundance normalized to miR-16-5p ( middle ), and the passenger strand (miR*) levels normalized to miR-16-5p ( right ). Values were normalized to expression level in sgNT for each transcript. n = 3 technical replicates per sgRNA, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test comparing sg1 or sg2 with sgNT. (***) P < 0.001, (****) P < 0.0001, (n.d.) not reliably detected.

Article Snippet: For E18.5 tissues, organs were homogenized in QIAzol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). cDNA was synthesized according to the manufacturer's instructions using Mir-X miRNA first strand synthesis kit (Takara) for miRNAs and U6 or PrimeScript RT (Takara) for all other transcripts. qRT-PCR was performed with SYBR Green Master mix (Applied Biosystems) on a QuantStudio 3 (Applied Biosystems). miR-16 or U6 snRNA was used to normalize qRT-PCR measurements of miRNA expression.

Techniques: Sequencing, Quantitative RT-PCR, Expressing, Infection, One-tailed Test

Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: Validation of novel TDMD triggers. ( A ) Schematic of trigger site deletion (Δts) strategy to validate putative TDMD triggers. ( B , D ) Genomic organization of TDMD trigger transcripts with conservation and predicted miRNA base-pairing architecture of the trigger sites. Nucleotides predicted to base-pair with the miRNA are shown in bold. ( C , E ) qRT-PCR analysis of indicated miRNAs, relative to miR-16-5p, in WT and Δts MEFs. Parental MEFs or two independent Δts clones for each trigger site were infected with lentivirus expressing Cas9 and a nontargeting CRISPR guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression level in sg Zswim8 for each condition. n = 3 technical replicates per clone, with individual data points plotted (mean ± SD shown). P -values were calculated by one-tailed Student's t -test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. ( F ) qRT-PCR analysis of the indicated miRNAs, relative to U6, in NIH3T3 cells at time points after serum starvation and restimulation. NIH3T3 cells expressing dCas9-KRAB were infected with lentivirus encoding a nontargeting guide (sgNT) or guides targeting Plagl1 or Lrrc58 . Parental NIH3T3 cells were infected with lentivirus expressing Cas9 and a nontargeting guide (sgNT) or Zswim8- targeting guide (sg Zswim8 ). Values were normalized to expression at the 0 h time point for each condition. n = 3 technical replicates per condition, with mean ± SD shown. P -values were calculated by one-tailed Student's t -test comparing sg Plagl1 (dCas9-KRAB) or sg Lrrc58 (dCas9-KRAB) with sgNT (dCas9-KRAB) or comparing sg Zswim8 (Cas9) with sgNT (Cas9). (*) P < 0.05.

Article Snippet: For E18.5 tissues, organs were homogenized in QIAzol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). cDNA was synthesized according to the manufacturer's instructions using Mir-X miRNA first strand synthesis kit (Takara) for miRNAs and U6 or PrimeScript RT (Takara) for all other transcripts. qRT-PCR was performed with SYBR Green Master mix (Applied Biosystems) on a QuantStudio 3 (Applied Biosystems). miR-16 or U6 snRNA was used to normalize qRT-PCR measurements of miRNA expression.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Clone Assay, Infection, Expressing, CRISPR, One-tailed Test

Journal: Genes & Development

Article Title: Plagl1 and Lrrc58 control mammalian body size by triggering target-directed microRNA degradation of miR-322 and miR-503

doi: 10.1101/gad.353138.125

Figure Lengend Snippet: Loss of the Plagl1 and Lrrc58 trigger sites abrogates TDMD of miR-322-5p and miR-503-5p in vivo. ( A ) Northern blot analysis of miRNA expression in E18.5 hearts ( left ) and lungs ( right ) in mice of the indicated genotypes. Quantification relative to miR-16-5p, normalized to mean expression in WT, is shown below each lane. ( B , C ) qRT-PCR analysis of miRNAs ( left ) and passenger strands ( right ) normalized to miR-16-5p in mouse tissues of the indicated genotypes at E18.5. Expression in all genotypes was normalized to mean expression in WT in each tissue. n = 3–6 mice per genotype, with each mouse represented by an individual data point (mean ± SD shown). P -values were calculated by one-tailed Student's t -test comparing Plagl1 Δ322ts ; Lrrc58 Δ503ts/Δ503ts with WT for each tissue. (Stom) Stomach, (Int) small intestine. (****) P < 0.0001. ( D ) Cumulative distribution function (CDF) plot showing the fold change in expression of the following sets of mRNAs in the brain ( left ) and lungs ( right ) at E18.5, comparing Plagl1 Δ322ts ; Lrrc58 Δ503ts/Δ503ts with WT: (1) all genes with counts per million (CPM) >5 (gray), (2) the set of conserved targets of miR-322-5p or miR-503-5p as predicted by TargetScan (light blue) , and (3) the set of conserved TargetScan-predicted targets that were also detected as chimeras with miR-322-5p or miR-503-5p in AGO-CLASH experiments (dark blue). P -values were calculated by one-sided Wilcoxon rank sum test.

Article Snippet: For E18.5 tissues, organs were homogenized in QIAzol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). cDNA was synthesized according to the manufacturer's instructions using Mir-X miRNA first strand synthesis kit (Takara) for miRNAs and U6 or PrimeScript RT (Takara) for all other transcripts. qRT-PCR was performed with SYBR Green Master mix (Applied Biosystems) on a QuantStudio 3 (Applied Biosystems). miR-16 or U6 snRNA was used to normalize qRT-PCR measurements of miRNA expression.

Techniques: In Vivo, Northern Blot, Expressing, Quantitative RT-PCR, One-tailed Test