Journal: The FASEB Journal

Article Title: Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear‐encoded noncoding RNAs

doi: 10.1096/fj.202401618R

Figure Lengend Snippet: List of RT qPCR primers.

Article Snippet: RNase treatment: To remove RNA not localized within intact mitochondria, in a 50 μL reaction, aliquots of isolated mitochondria (1 μg total protein each) first had 100 ng mRNA spike‐in control added (StemMACS eGFP mRNA #130‐101‐114, Miltenyi Biotec) and were then incubated with or without Triton X‐100 (1% v/v) and with or without RNase‐A (Qiagen #19101, final concentration range 1–1000 μg/mL) and incubated on ice for 30 min. TRIzol reagent was then added to simultaneously inactivate RNase activity, lyse mitochondria, and preserve RNA.

Techniques: Quantitative RT-PCR

Journal: The FASEB Journal

Article Title: Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear‐encoded noncoding RNAs

doi: 10.1096/fj.202401618R

Figure Lengend Snippet: RNase‐A treatment of mitochondria isolated from L6 myocytes. Isolated mitochondria were incubated with or without RNase‐A (1–1000 μg/mL) in the presence or absence of detergent (1% Triton X‐100). The levels of representative (A) mtDNA ( Mt‐co3 )‐ and (B) nuclear ( Cox4i1 )‐encoded genes were then assessed by RT‐qPCR in addition to (C) an exogenous mRNA spike‐in control ( eGFP ) added to isolated mitochondria (but not to the whole cell lysate). The values were derived by exponential transformation of the C q value, and data for each gene are expressed as a fraction of the untreated isolated mitochondria condition (relative expression). Data are mean (SD) for N = 3 technical replicates from a single mitochondrial isolation preparation.

Article Snippet: RNase treatment: To remove RNA not localized within intact mitochondria, in a 50 μL reaction, aliquots of isolated mitochondria (1 μg total protein each) first had 100 ng mRNA spike‐in control added (StemMACS eGFP mRNA #130‐101‐114, Miltenyi Biotec) and were then incubated with or without Triton X‐100 (1% v/v) and with or without RNase‐A (Qiagen #19101, final concentration range 1–1000 μg/mL) and incubated on ice for 30 min. TRIzol reagent was then added to simultaneously inactivate RNase activity, lyse mitochondria, and preserve RNA.

Techniques: Isolation, Incubation, Quantitative RT-PCR, Control, Derivative Assay, Transformation Assay, Expressing

Journal: Cell Reports

Article Title: Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches

doi: 10.1016/j.celrep.2021.109618

Figure Lengend Snippet: Osteo-angiogenic development of long bones is disrupted in VEGF cTg mice (A) Total VEGF mRNA (qRT-PCR) and VEGF 164 protein levels (ELISA) in bones of control and VEGF cTg embryos (n = 4–8). (B) VEGF serum levels (ELISA) (n = 6). (C) Skeletal preparations at E18.5. Middle panels, dissected hindlimbs of control (top) and VEGF cTg (bottom). (D) Longitudinal (top) and transversal (bottom) 3D micro-computed tomography (micro-CT) reconstructions of E18.5 tibias. (E) Histology of E18.5 tibias; (left) Safranin O and (right) H&E. Brackets, BM cavity. (F) BM cavity volume at E18.5 (n = 7). (G) CD31 IHC on tibia sections. Arrows, aberrant perichondrial vascularization. (H) Maximum intensity projection (MIP) of 20-μm depth confocal imaging of Osx-Cre:GFP (nuclear green, OPCs), Emcn (red, blood vessels), and Hoechst (blue, nuclei) in E18.5 tibias. Left, overview; middle, metaphysis; right, diaphysis. (I) Flow cytometry of CD45 − Ter119 − CD31 − Osx/GFP + cells in E18.5 bones (n = 4–5). All graphs represent mean ± SEM and depict individual data points derived from different mice (also in the next figures); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, by Student’s t test between the genotypes for each time point; no symbol implies p > 0.05. See also Figure S1 .

Article Snippet: The extraction of total RNA (RNeasy Micro kit, QIAGEN), library preparation using 100 ng RNA (TruSeq Stranded mRNA Sample Preparation Kit, Illumina), indexing, quantification, validation of library quality and size range (DNA 1000 kit, Agilent Technologies), sequencing Illumina HiSeq2500, generating 50 bp single-end reads with a depth of 30-40 Mb per sample), and processing of results and bioinformatics was all done according to the procedures described before ( ).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control, Micro-CT, Imaging, Flow Cytometry, Derivative Assay

Journal: Cell Reports

Article Title: Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches

doi: 10.1016/j.celrep.2021.109618

Figure Lengend Snippet: Delayed-onset VEGF overexpression in developing bones creates a hypomorph model of osteo-angiogenic alterations with a normal-size BM cavity (A) Total VEGF mRNA and VEGF 164 protein levels at E18.5 and P2 (n = 6–11). (B) VEGF serum levels at E18.5 (n = 6). (C) E18.5 skeletal preparations; bottom, hindlimbs. (D) H&E and BM cavity length of E18.5 tibias (n = 3). (E–G) Micro-CT of E18.5 tibias with (F) bone volume/tissue volume (BV/TV) and (G) BM cavity volume (n = 5). (H and I) CD31 IHC and (I) histomorphometry on E18.5 tibias. BLV, blood vessel (n = 3–4). (J) MIP (15-μm depth) of Osx--Cre:GFP, Emcn, and Hoechst signals in P2 tibias. (K) Flow cytometry of CD45 − Ter119 − CD31 − Osx-Cre:GFP + cells in P2 limbs (n = 7). (L and M) LSK, LSK-SLAM, and CD45 + cell counts in (L) bones and (M) spleens (n = 3–8). Graphs, mean ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, t test. See also Figure S3 .

Article Snippet: The extraction of total RNA (RNeasy Micro kit, QIAGEN), library preparation using 100 ng RNA (TruSeq Stranded mRNA Sample Preparation Kit, Illumina), indexing, quantification, validation of library quality and size range (DNA 1000 kit, Agilent Technologies), sequencing Illumina HiSeq2500, generating 50 bp single-end reads with a depth of 30-40 Mb per sample), and processing of results and bioinformatics was all done according to the procedures described before ( ).

Techniques: Over Expression, Micro-CT, Flow Cytometry

Journal: Cell Reports

Article Title: Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches

doi: 10.1016/j.celrep.2021.109618

Figure Lengend Snippet:

Article Snippet: The extraction of total RNA (RNeasy Micro kit, QIAGEN), library preparation using 100 ng RNA (TruSeq Stranded mRNA Sample Preparation Kit, Illumina), indexing, quantification, validation of library quality and size range (DNA 1000 kit, Agilent Technologies), sequencing Illumina HiSeq2500, generating 50 bp single-end reads with a depth of 30-40 Mb per sample), and processing of results and bioinformatics was all done according to the procedures described before ( ).

Techniques: Flow Cytometry, Immunohistochemistry, Recombinant, Staining, Extraction, Modification, Electron Microscopy, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Purification, Reverse Transcription, RNA Sequencing, Control, Software

Journal: Nature medicine

Article Title: TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma

doi: 10.1038/s41591-020-0757-z

Figure Lengend Snippet: a, A screenshot of a representative sequence trace file of a CRISPR-generated TMEM30A frameshift mutation in exon 1 (DOHH-2, MUT1). b, TMEM30A mRNA expression is reduced in 3 unique bi-allelic frame-shift mutations in Karpas422, compared with parental and single cell enriched controls. mRNA expression is normalized to GAPDH expression. Graphs represent mean values ± s.d. (n = 3). (c) mRNA expression of TMEM30A is ‘rescued’ in one unique bi-allelic TMEM30A mutant, by ectopically expressing a stable insertion of a TMEM30A cDNA. Graphs represent mean values ± s.d. (n = 3). d, mRNA levels of different scramblases (PLSCR1-5; ANO1-10, XKR1-9), flippases (TMEM30A-C) and P4-ATPases in Karpas422 (left panel) and NUDUL-1 (right panel) native (WT, CON) and TMEM30A−/− (MUT) cell lines. Protein family members are categorized, and the heat map values show log-normalized expression by RNA-seq. e, XKR8 and ANO6 mRNA expression is reduced in 3 unique bi-allelic (XKR8) and mono-allelic (ANO6) frame-shift mutations in TMEM30A−/− and wildtype controls. mRNA expression is normalized to GAPDH expression. Graphs represent mean values ± s.d. (n = 3). All P values are based on a paired two-tailed Student’s t test.

Article Snippet: Polyadenylated (polyA+) mRNA was purified using the 96-well MultiMACS mRNA isolation kit on the MultiMACS 96 separator (Miltenyi Biotec) then ethanol-precipitated, and used to synthesize complementary DNA using the Maxima H Minus First Strand cDNA Synthesis kit (Thermo-Fisher) and random hexamer primers at a concentration of 5 μM along with a final concentration of 1 μg μl −1 Actinomycin D, followed by Ampure XP SPRI bead purification on a Biomek FX robot (Beckman-Coulter).

Techniques: Expressing, In Vitro, Knock-Out, Sequencing, CRISPR, Generated, Mutagenesis, RNA Sequencing, Two Tailed Test