Fig 1.
Non-normalized expression profiles (2-ΔCq) of reference genes derived from RNA-Seq data of iPSC derived microglia.
WT group was used as the experimental calibrator. (A) ANXA7, (B) APIP, (C) CCNT2, (D) FOXK1, (E) KIF13A, (F) USP5, (G) CNBP, (H) DDX42, (I) MRPL37, (J) PPP1R10 and (K) Normalization Factor (NF) assessed by NormFinder that combines CNBP & KIF13A. Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05.
Table 1.
CV and NormFinder analysis RNA-Seq derived reference genes in iPSC microglia.
Expression stability of candidate reference genes derived from bulk RNA-Seq data were evaluated using Coefficient of Variation (CV) Analysis & NormFinder.
Fig 2.
Non-normalized expression profiles (2-ΔCq) of conventional human reference genes in iPSC derived microglia.
WT group is used as the experimental calibrator. (A) ACTB, (B) GAPDH, (C) GUSB, (D) PPIA, (E) RPLP0, (F) TBP, (G) HPRT, (H) PGK1, (I) UBC, (J) RPL13A, and (K) Normalization Factor (NF) assessed by NormFinder that combines RPLP0 & GUSB. Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05, ** P<0.01, *** P<0.001.
Table 2.
CV and NormFinder analysis of conventional reference genes iPSC microglia.
Expression stability of conventional candidate reference genes were evaluated using Coefficient of Variation (CV) Analysis, NormFinder.
Fig 3.
Differential expression of target genes between WT and TREM2KO iPSC cells assessed by qPCRs and RNA-Seq.
The target genes (TREM2, P2RY12, CD52, ACSS3, SYDE1, TBX10) have been normalized with the normalization factor (NF) computed from conventional reference genes RPLP0 & GUSB in the first column (A, D, G, J, M, P) or from RNA-Seq derived reference genes CNBP & KIF13A in the second column (B, E, H, K, N, Q). The third column contains the Fold Changes computed from RNA-Seq (C, F, I, L, O, R). The normalized counts for each gene in each group were used to calculate the fold changes in RNA-Seq. The WT group was used as the experimental control. The Padj value of RNA-Seq is indicated in the TREM2KO group for each gene in the RNA-Seq column. For the qPCRs, Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05.
Fig 4.
Non-normalized expression profiles (2-ΔCq) of reference genes derived from RNA-Seq analysis of sciatic nerves at post-natal day 3 (P3) and 21 (P21).
P3 group is the experimental calibrator. (A) Fkbp4, (B) Lama2, (C) Leprotl1, (D) Supt4a, (E) Coq9, (F) Chmp2a, (G) Ank2, (H) Vcp, (I) Laptm5, (J) Ppp3ca, and (K) Normalisation Factor (Ppp3ca + Coq9). Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05, ** P<0.01, *** P<0.001.
Table 3.
CV and NormFinder analysis of reference genes derived from RNA-Seq in sciatic nerves.
Fig 5.
Non-normalized expression profiles (2-ΔCq) of conventional reference genes of sciatic nerves at post-natal day 3 (P3) and 21 (P21).
P3 group is the experimental calibrator. (A) Actb, (B) Mrpl10, (C) Hsp60, (D) Ppia, (E) Rpl13a, (F) Tbp, (G) Gapdh, (H) Pgk1, (I) Rps26, (J) Sdha, and (K) Normalisation Factor (Hsp60 + Ppia). Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05, ** P<0.01, *** P<0.001.
Table 4.
CV and NormFinder analysis of conventional reference genes in sciatic nerves.
Fig 6.
Differential expression of target genes between P3 and P21 mouse sciatic nerves assessed by qPCRs and RNA-Seq.
The target genes (Mki67, p75NTR, Sox2, Mbp, Mpz, Cd90) have been normalized with the normalization factor (NF) computed from conventional reference genes Hsp60 & Ppia in the first column (A, D, G, J, M, P) or from RNA-Seq derived reference genes Ppp3ca and Coq9 in the second column (B, E, H, K, N, Q). The third column contains the Fold Changes computed from RNA-Seq (C,F,I,L,O,R). The normalized counts for each gene in each group were used to calculate the fold changes in RNA-Seq. The WT group was used as the experimental control. The Padj value of RNA-Seq is indicated in the P21 group for each gene in the RNA-Seq column. For the qPCRs, Non-parametric Mann Whitney U test was used to assess differences between the groups. The alpha value was set at 0.05 and P values are annotated as follows: * P<0.05, ** P<0.01, *** P<0.001.