exogenous rna control  (TaKaRa)

Journal: Nucleic Acids Research

Article Title: Nuclear poly(A) binding protein 1 (PABPN1) mediates zygotic genome activation-dependent maternal mRNA clearance during mouse early embryonic development

doi: 10.1093/nar/gkab1213

Figure Lengend Snippet: Transcriptome analyses in Pabpn1 -depleted embryos during the MZT. ( A ) Schematic diagram showing the samples used for RNA-seq, circled by a dashed line. Zygotes, late 2-cell, and 8-cell embryos were collected from in vivo at 28, 43 and 72 h post-hCG injection. The red represents the subcellular localization and expression level of PABPN1. ( B ) Changes in relative mRNA copy numbers in control and Pabpn1 -depleted embryos during the zygote-to-2-cell transition. Total mRNA copy numbers were calculated by normalizing with the ERCC spike-in, and mRNA expression at the zygote stage was set to 1.0. ** P < 0.01 by two-tailed Student's t -test. ( C ) Classification of transcripts according to the expression pattern changes during the zygote-to-2-cell transition in WT embryos into three groups. Group 1: FPKM (zygote/2-cell) > 2; Group 2: FPKM (2-cell/zygote) > 2; Group 3: FPKM (zygote/2-cell) < 2 and FPKM (2-cell/zygote) <2. Each light blue line represents the expression level of one gene in WT embryos. The middle blue line and red line indicate the median expression level of the group in WT and si Pabpn1 embryos, respectively. ( D ) Scatter plot showing changes in transcript levels in 2-cell embryos after Pabpn1 depletion. Transcripts whose levels increased or decreased by more than 2-fold in Pabpn1 -depleted embryos are highlighted in red or blue, respectively. ( E ) Venn diagrams showing the overlap of the upregulated transcripts in Pabpn1 -depleted embryos (FPKM (si Pabpn1 /WT) > 2 in 2-cell embryos) and the degraded transcripts from the zygote to 2-cell embryos in WT (FPKM (zygote/2-cell) > 3 in WT), as well as the upregulated transcripts in Tut4/7 -depleted embryos (FPKM (si Tut4/7 /WT) > 2 in 2-cell embryos). P = 1e-1753 by two-tailed Student's t-test. ( F ) RT-qPCR results showing the relative mRNA levels of the indicated Z-decay transcripts in 2-cell embryos with or without Pabpn1 depletion. Error bars, S.E.M. * P < 0.05, ** P < 0.01 by two-tailed Student's t -test. n = 3 biological replicates. ( G ) Venn diagram showing the overlap in transcripts whose levels increased during the zygote-to-2-cell transition in WT (FPKM (2-cell/zygote) > 2) and those whose levels decreased in Pabpn1 -depleted embryos compared with those in WT at the 2-cell stage (FPKM (WT/si Pabpn1 > 2 in 2-cell embryos), as well as transcripts whose levels decreased in Tut4/7 -depleted embryos (FPKM (WT/si Tut4/7 ) > 2 in 2-cell embryos). P = 1e–1531 by two-tailed Student's t -test. ( H ) RT-qPCR results showing relative levels for the indicated zygotic genome activation (ZGA) transcripts in 2-cell embryos with or without Pabpn1 depletion. Error bars, S.E.M. * P < 0.05, *** P < 0.001 by two-tailed Student's t -test. n = 3 biological replicates.

Article Snippet: The expression level of each gene was quantified and indicated as normalized FPKM based on the FPKM of exogenous External RNA Controls Consortium (ERCC) (Invitrogen, Cat. No. 4456740) transcript mixtures.

Techniques: RNA Sequencing Assay, In Vivo, Injection, Expressing, Two Tailed Test, Quantitative RT-PCR, Activation Assay