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: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in FACS‐isolated Lin(Ter119/CD31/CD45) − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 1 μM cPGI 2 or vehicle for the indicated time, as determined by expression profiling ( n = 3, E‐MTAB‐3693). **** P = 3 × 10 −6 (Day 2), **** P = 4 × 10 −7 (Day 4), **** P = 1 × 10 −6 (Day 6) in 2 × 2 ANOVA with Bonferroni's posttests (cPGI 2 vs. vehicle). B mRNA expression in MACS‐isolated Lin − Sca1 + progenitor cells from female mouse subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 4). **** P = 1 × 10 −10 (Days 1 and 2), ** P = 0.001, in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). C mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi) * P = 0.013 ( Ucp1 ), ** P = 0.004 ( Cpt1b ), * P = 0.026 ( Dio2 ). D–F mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi (D) or vehicle (D–F), as determined by qRT–PCR at the indicated time points ( n = 5 patients). ♀/♂ represents individual data. (D) **** P = 3 × 10 −5 (Day 2), **** P = 3 × 10 −6 (Day 6), **** P = 4 × 10 −9 (Day 10), **** P = 3 × 10 −7 (Day 14), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). (E, F) Pearson correlation coefficient ( r ) and P ‐value are shown. G mRNA expression in primary SVF cells from human female subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( CITED4 ), ** P = 0.002 ( UCP1 ), * P = 0.02 ( UCP1 ), * P = 0.035/0.026 ( PPARG ), *** P = 0.0006 ( SLC2A4 ), ** P = 0.002 ( ADIPOQ ) in one‐way ANOVA with Tukey's posttests (vs. siCtrl ). Data information: Data are presented as mean ± SEM except for (D) ♀/♂, (E and F) individual data.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Transfection

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in C3H10T1/2 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR (day 0, n = 3; day 4, n = 4; day 6, Ctrl, n = 2, Rosi, n = 4; day 10, n = 4). *** P = 0.001, ** P = 0.007 (Day 6), ** P = 0.005 (Day 10), in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). B mRNA expression in 3T3‐L1 cells, differentiated in the presence of 1 μM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 3). 2 × 2 ANOVA with Bonferroni's posttests, P > 0.05 (Rosi vs. vehicle). C mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of 0.1 or 1 μM pioglitazone (Pio) or vehicle for 8 days, as determined by qRT–PCR ( n = 4 for Day 2, n = 4/2/6 for Day 8). *** P = 1*10 −10 , ** P = 0.003, in 2 × 2 ANOVA with Bonferroni's posttests (Pio vs. vehicle). D mRNA expression in female Lin − Sca1 + cells, differentiated in the presence of the indicated substances for 8 days, as determined by qRT–PCR ( n = 3). ** P = 0.0035, *** P = 4 × 10 −10 , *** P = 7 × 10 −10 ( Ucp1 ), * P = 0.036, *** P = 0.0002, *** P = 1 × 10 −10 , *** P = 1 × 10 −10 ( Cpt1b ), *** P = 2 × 10 −5 ( Cidea ), *** P = 1 × 10 −6 , *** P = 4 × 10 −9 , *** P = 1 × 10 −8 , ( Elovl3 ), *** P = 9 × 10 −8 , *** P = 2 × 10 −7 ( Cox7a1 ), *** P = 0.0002, *** P = 1 × 10 −8 , *** P = 4 × 10 −5 , *** P = 0.0002 ( Cox8b ), * P = 0.032, ** P = 0.006, *** P = 0.0007 ( Dio2 ), *** P = 1 × 10 −6 , *** P = 2 × 10 −8 ( Cyc1 ), *** P = 8 × 10 −5 , *** P = 3 × 10 −6 ( Ndufb3 ) in 2 × 2 ANOVA with Holm–Sidak posttests ( Cited4 −/− vs. Cited4 +/+ ). E mRNA expression in male Lin − Sca1 + progenitor cells, differentiated in the presence of 100 nM Rosi or vehicle for the indicated time, as determined by qRT–PCR ( n = 3). **** P = 1 × 10 −10 (Days 2 and 4), **** P = 4 × 10 −8 (Day 8) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). F mRNA expression in male Lin − Sca1 + cells, differentiated in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3, t ‐test). Data information: Data are presented as mean ± SEM except for (C) (individual data).

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A Phase contrast microscopy of primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle for 14 days (representative of n = 5 patients). Scale bar is 100 μm. B, C mRNA expression in primary SVF cells from human subcutaneous fat, differentiated in the presence of 100 nM Rosi or vehicle, as determined by qRT–PCR ( n = 5 patients). (B) **** P = 1 × 10 −10 (Day 2), **** P = 5 × 10 −8 (Day 6), **** P = 1 × 10 −9 (Days 10 and 14), (C) **** P = 7 × 10 −7 (Day 6), **** P = 1 × 10 −9 (Days 10 and 14) in 2 × 2 ANOVA with Bonferroni's posttests (Rosi vs. vehicle). D mRNA expression in primary SVF cells from human male subcutaneous fat transfected with the indicated siRNA prior to differentiation in the presence of 100 nM Rosi for 9 days, as determined by qRT–PCR ( n = 3). *** P = 0.0002 ( siCITED4.1 CITED4 ), *** P = 0.0003 ( siCITED4.2 CITED4 ), ** P = 0.005 ( UCP1 ), * P = 0.01 ( CIDEA ), *** P = 0.0006 ( CITED4.1 CPT1B ), ** P = 0.007 ( CPT1B ), *** P = 0.0006 ( CITED4.1 SLC2A4 ),** P = 0.009 ( ADIPOQ ) in ANOVA with Tukey's posttests (vs. siCtrl ). E GFP fluorescence intensity distribution of Lin − Sca1 + progenitor cells 24 hours after transfection with GFP mRNA, determined by flow cytometry (compared to non‐transfected cells). F mRNA expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 3). t ‐test Cre vs. Ctrl (Rosi), ** P = 0.002 ( Cited4 ), * P = 0.039 ( Adipoq ), * P = 0.015 ( Ucp1 ), * P = 0.011 ( Cidea ), ** P = 0.004 ( Cpt1b ), * P = 0.011 ( Dio2 ). G mRNA expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA 3 days after induction of differentiation in the presence of 100 nM Rosi or vehicle for 8 days, as determined by qRT–PCR ( n = 4). t ‐test Cre vs. Ctrl, *** P = 0.0005 ( Cited4 ), ** P = 0.009 ( Cpt1b ). Data information: Data are presented as mean ± SEM except for (E) (individual data).

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Microscopy, Expressing, Quantitative RT-PCR, Transfection, Fluorescence, Flow Cytometry, Control

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A–C Quantitative fluorescence microscopy of LipidTOX‐ and DAPI‐stained female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days ( n = 5). ** P = 0.002 in t ‐test (Cre vs. Ctrl). Scale bar is 10 μm. D, E Ucp1 expression in female Cited4 F/F Lin − Sca1 + progenitor cells transfected with Cre or control mRNA prior to differentiation in the presence of 100 nM Rosi for 8 days, as determined by Western blot with VCP as loading control ( n = 3). *** P = 0.0008 in t ‐test (Cre vs. Ctrl). F Cellular respiration in female Lin − Sca1 + progenitor cells differentiated in the presence of 100 nM Rosi for 8 days. The extracellular oxygen consumption rate (OCR) was determined upon injection of the indicated substances and normalized to DNA content. CL: CL‐316243. Representative experiment ( n = 9). * P = 0.041 (47 min), ** P = 0.009 (55 min), ** P = 0.003 (62 min) in 2 × 2 ANOVA with Holm–Sidak posttests ( Cited4 −/− vs. Cited4 +/+ ). G Normalized CL‐stimulated uncoupled respiration of cells in (F). Values represent the means of the three time points after CL injection after subtraction of the means of the oligomycin time points ( n = 9). * P = 0.027 in t ‐test ( Cited4 −/− vs. Cited4 +/+ ). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Fluorescence, Microscopy, Staining, Transfection, Control, Expressing, Western Blot, Injection

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A mRNA expression in wild‐type mice without treatment, as determined by qRT–PCR ( n = 6). scWAT: subcutaneous white adipose tissue; gWAT: gonadal WAT; isBAT: interscapular brown adipose tissue; SKM: gastrocnemius skeletal muscle. 2 × 2 ANOVA with Holm–Sidak posttests, *** P = 0.0004 (female vs. male), ### P = 0.0004, ### P = 7 × 10 −6 , ### P = 6 × 10 −9 , ### P = 3 × 10 −10 , (isBAT, SKM, heart, liver, respectively, vs. scWAT within indicated sex), # P = 0.032 (females: gWAT vs. scWAT), ## P = 0.006 (females: Heart vs. scWAT), ## P = 0.006 (males: Liver vs. scWAT). B Ucp1 expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by Western blot with VCP as loading control ( n = 3, same samples as in Fig C and D). C mRNA expression in BAT of mice treated as in (A), determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, males: n = 5/4/5/5, *** P = 0.0004. D mRNA expression in gWAT of mice treated as in (A), determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, males: n = 5/4/5/5. E Three‐day averages of total activity counts per day and mouse of female mice fed a diet with 0.0075% Rosi for 2.5 weeks, determined by Phenomaster ( n = 9/10, t ‐test). F Three‐day averages of food intake per day and mouse of female mice shown in (E) ( n = 9/10, t ‐test). G Oxygen consumption rate of female mice fed control diet, determined by indirect calorimetry and adjusted for body weight by ANCOVA. Three‐day averages of VO 2 were calculated for each mouse ( n = 10). ANCOVA with Bonferroni's posttest ( Cited4 −/− vs. Cited4 +/+ ). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A, B mRNA expression in scWAT of mice fed a diet with 0.0075% Rosi or control diet for 2.5 weeks, determined by qRT–PCR. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), (A) n = 5/5/6/6, ** P = 0.003 ( Ucp1 ), ** P = 0.008 ( Cidea ), ** P = 0.007 ( Cyc1 ), * P = 0.048 ( Ndufb3 ), (B) n = 5/4/5/5, * P = 0.022 ( Ucp1 ), * P = 0.037 ( Cidea ). C, D Ucp1 expression in scWAT of mice treated as in (A), as determined by Western blot with VCP as loading control. t ‐test Cited4 −/− vs. Cited4 +/+ (Rosi), females: n = 5/5/6/6, * P = 0.011, males: n = 5/4/5/5. E mRNA expression in scWAT of female mice treated with CL‐316,243 (CL) (1 mg/kg/day via Alzet minipumps) or vehicle for 10 days ( n = 5/7/13, t ‐test Cited4 −/− vs. Cited4 +/+ (CL)). F mRNA expression in scWAT of female mice exposed to 5°C or 23°C for 2 weeks ( n = 5/10/6, t ‐test Cited4 −/− vs. Cited4 +/+ (5°C)). Data information: Data are presented as mean ± SEM. Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Cited4 is a sex‐biased mediator of the antidiabetic glitazone response in adipocyte progenitors

doi: 10.15252/emmm.201708613

Figure Lengend Snippet: A, B mRNA expression in scWAT of mice fed a high‐fat diet (HFD) for 11 weeks followed by 5 weeks of HFD with 0.0075% Rosi (qRT–PCR). t ‐test Cited4 −/− vs. Cited4 +/+ , (A) n = 8/7, * P = 0.029 ( Ucp1 ), ** P = 0.009 ( Elovl3 ), ** P = 0.006 ( Cpt1b ), ** P = 0.004 ( Cyc1 ), * P = 0.016 ( Ndufb3 ), ** P = 0.003 ( Cd36 ), ** P = 0.009 ( Gyk ), (B) n = 7/8, * P = 0.026 ( Pck1 ). C, D Ucp1 protein expression in scWAT of mice treated as in (A), determined by Western blot with VCP as loading control ( n = 8/7 for females and n = 7/8 for males). Arrow indicates the band specific to Ucp1 (see Appendix Fig S5F ). Data information: Data presented as mean ± SEM (A and B), individual mice (D, males), or means of replicate blots with independent lysates from the same mice (D, females). Source data are available online for this figure.

Article Snippet: For the transfection of primary Lin − Sca1 + cells from Cited4 F/F mice with StemMACSTM Cre recombinase (130‐101‐113; Miltenyi Biotec) or StemMACSTM Nuclear eGFP messenger RNA (mRNA) (130‐101‐119; Miltenyi Biotec), a mixture of Lipofectamine ® RNAiMAX transfection reagent (2 μl/ml) and Opti‐MEM medium was combined with Opti‐MEM containing the respective mRNA (500 ng/ml final concentration).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Advanced Science

Article Title: A General and Efficient Strategy for Gene Delivery Based on Tea Polyphenols Intercalation and Self‐Polymerization

doi: 10.1002/advs.202302620

Figure Lengend Snippet: TPNs as a versatile gene vector to package various types of nuclei acids. A) Gel images and intensity quantification to show the loading capacity of TPNs toward ASO, siRNA, mRNA, and pDNA at different feeding concentrations. B,D) The particle size and C,E) ζ potential of ASO@TPN, siRNA@TPNs, mRNA@TPNs, and pDNA@TPNs prepared at different concentrations ( n = 3). F) The size distribution of TPNs, ASO@TPNs, siRNA@TPNs, mRNA@TPNs, and pDNA@TPNs. G) The gel image and gray scale values to show the adsorption of ASO and siRNA on TPNs surface at different feeding concentrations ( n = 3). Data were presented as mean ± SD.

Article Snippet: ARCA‐EGFP mRNA was purchased from APExBIO (Houston, USA).

Techniques: Plasmid Preparation, Adsorption

Journal: Advanced Science

Article Title: A General and Efficient Strategy for Gene Delivery Based on Tea Polyphenols Intercalation and Self‐Polymerization

doi: 10.1002/advs.202302620

Figure Lengend Snippet: In vitro transfection of various nuclei acids by TPNs. A) The fluorescence images of RAW264.7 cells incubated with ASO/siRNA (8 h) or mRNA/pDNA (36 h) in the naked form or encapsulated by TPNs. Scale bar = 100 µm. The flow cytometry to quantify the cellular internalization of B) ASO@TPNs, C) siRNA@TPNs, D) mRNA@TPNs, and E) pDNA@TPNs after incubating for different timepoints ( n = 3). F) The mRNA levels and G) protein levels of TNF‐ α in RAW264.7 cells post different treatments ( n = 3). H) The cellular internalization of siRNA@TPNs with pre‐treatment of various endocytosis inhibitors ( n = 3). I) The intracellular co‐localization of siRNA@TPNs with lysosomes after incubating for different timepoints. Scale bar = 20 µm. Data were presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA for (B–H). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ARCA‐EGFP mRNA was purchased from APExBIO (Houston, USA).

Techniques: In Vitro, Transfection, Fluorescence, Incubation, Flow Cytometry

Journal: Advanced Science

Article Title: A General and Efficient Strategy for Gene Delivery Based on Tea Polyphenols Intercalation and Self‐Polymerization

doi: 10.1002/advs.202302620

Figure Lengend Snippet: Antioxidation, anti‐inflammation, and macrophage repolarization effects of TPNs vector. A) The fluorescence images of RAW264.7 cells with different treatments showing the capacity of siRNA@TPNs to scavenge RONS. Scale bar = 100 µm. The flow cytometry results of TPNs to scavenge B) general ROS, C) ·O 2 − , D) ·OH/ONOO − , and E) ·NO ( n = 3). F) Immunofluorescence images of iNOS and CD206 in RAW264.7 cells with different treatments. Scale bar = 100 µm. The flow cytometry results of G) iNOS and H) CD206 expression in RAW264.7 cells with different treatments ( n = 3). I) The mRNA levels and J) protein levels of TNF‐ α , IL‐1 β , and IL‐6 in RAW264.7 cells post different treatments ( n = 3). Data were presented as mean ± SD. Statistical comparisons were performed using one‐way ANOVA for (B–E) and (G–J). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ARCA‐EGFP mRNA was purchased from APExBIO (Houston, USA).

Techniques: Plasmid Preparation, Fluorescence, Flow Cytometry, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: mRNA vaccine quality analysis using RNA sequencing

doi: 10.1038/s41467-023-41354-y

Figure Lengend Snippet: Schematic diagram illustrates the steps during mRNA manufacture (left panel), and the steps during VAX-seq analysis (right panel). This includes laboratory steps of long-read nanopore sequencing, followed by bioinformatic steps to analyse output data, including the supporting Mana software toolkit. mRNA vaccine quality features that can be analysed by VAX-seq are indicated (listed in red and green). In the bottom left corner is an IGV plot comparing Oxford Nanopore and Illumina sequencing of a plasmid DNA template. Coverage indicates the number of reads at each nucleotide position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. Source data are provided as a Source data file.

Article Snippet: Fig. 2 Analysis of reference eGFP mRNA vaccine using long-read Oxford Nanopore sequencing (PCS111). a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence.

Techniques: Nanopore Sequencing, Software, Illumina Sequencing, Plasmid Preparation

Journal: Nature Communications

Article Title: mRNA vaccine quality analysis using RNA sequencing

doi: 10.1038/s41467-023-41354-y

Figure Lengend Snippet: a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence. Coverage indicates the number of reads at each nucleotide position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. b Sequencing error and type profile across mRNA vaccine and poly(A) tail sequences. c Detail shows sequencing coverage and error at the poly(A) tail, showing a characteristic m-shaped deletion profile. d mRNA length as measured using ONT full-length sequencing shows full-length and fragmented mRNA vaccines. e poly(A) tail length measured using tailfindr for eGFP mRNA (green), compared to a cDNA library with variable poly(A) tail lengths. Source data are provided as a Source data file.

Article Snippet: Fig. 2 Analysis of reference eGFP mRNA vaccine using long-read Oxford Nanopore sequencing (PCS111). a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence.

Techniques: Plasmid Preparation, Sequencing, Vaccines, cDNA Library Assay

Journal: Nature Communications

Article Title: mRNA vaccine quality analysis using RNA sequencing

doi: 10.1038/s41467-023-41354-y

Figure Lengend Snippet: a Genome-browser (IGV) view of short-read cDNA sequenced libraries aligned to the reference plasmid sequence. Coverage indicates the alignment depth at each nucleotide position, while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. b Detail of transcription start site shows alignment of long- and short-read sequencing at 5’ end of mRNA vaccine. c Detail genome browser view of poly(A) tail shows uneven coverage and deletion of linker sequence due to short-read misalignment. Source data are provided as a Source data file.

Article Snippet: Fig. 2 Analysis of reference eGFP mRNA vaccine using long-read Oxford Nanopore sequencing (PCS111). a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence.

Techniques: Plasmid Preparation, Sequencing

Journal: Nature Communications

Article Title: mRNA vaccine quality analysis using RNA sequencing

doi: 10.1038/s41467-023-41354-y

Figure Lengend Snippet: a Genome-browser (IGV) view of direct RNA sequencing alignments to the reference sequence. Coverage indicates the number of reads at each nucleoside position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. b Direct RNA sequencing shows error type and frequencies across the mRNA vaccine and poly(A) tail sequence. c Plot shows the length of mRNA vaccine as measured from direct RNA sequencing, showing additional smaller peak resulting from artefactual trimming of poly(A) tail from reads. d Poly(A) tail length measured using tailfindr across three technical replicate direct RNA sequencing libraries. e Schematic diagram shows the different mRNA species, fragment size and contaminations identified by the VAX-seq workflow. Source data are provided as a Source data file.

Article Snippet: Fig. 2 Analysis of reference eGFP mRNA vaccine using long-read Oxford Nanopore sequencing (PCS111). a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence.

Techniques: RNA Sequencing, Sequencing

Journal: Nature Communications

Article Title: mRNA vaccine quality analysis using RNA sequencing

doi: 10.1038/s41467-023-41354-y

Figure Lengend Snippet: a , b Genome-browser (IGV) view of long-read (ONT) alignment to the plasmid reference, sequenced using direct RNA sequencing for mRNA vaccines prepared with a Uridine and b N1 methylpseudouridine. Coverage indicates the number of reads at each nucleoside position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. Heterogenous coverage is observed in the direct RNA sequencing, likely due to fragmentation of the modified mRNA vaccine. c mRNA length analyses demonstrate shorter length for modified mRNA vaccines due to fragmentation and enrichment in deletion sequencing errors ( n = 4). d Direct RNA sequencing shows stereotypical errors (cytosine, blue; uridine; red) at N1-methylpseudouridine compared to uridine nucleosides. e Cumulative distribution plot shows per-nucleoside error profile for N1-methylpseudouridine (red/orange) compared to unmodified nucleosides (blue/green). Source data are provided as a Source data file.

Article Snippet: Fig. 2 Analysis of reference eGFP mRNA vaccine using long-read Oxford Nanopore sequencing (PCS111). a Genome-browser (IGV) view of long-read cDNA alignments to the reference plasmid sequence.

Techniques: Plasmid Preparation, RNA Sequencing, Vaccines, Modification, Sequencing

Journal: bioRxiv

Article Title: High throughput intracellular delivery by viscoelastic mechanoporation

doi: 10.1101/2023.04.24.538131

Figure Lengend Snippet: (a) eGFP expression 24 hours after mRNA transfection to Jurkat cells for a range of extracellular mRNA concentrations. (b) T cell receptor (TCR) expression in Jurkat cells 2 days after transfection of Cas9 RNP complexes designed to knock out the TCR, for a range of RNP concentrations ( n = 3 for experimental conditions, n = 1 for controls). (c) Viability and delivery of 70 kDa FITC-dextran to HEK293T cells, 24 hours after processing, for three delivery buffer compositions and 2 different chip pressures (Unstretched, n = 2 ; PBS, n = 1 ; Cytomix buffers, n = 3 ). (d) Viability and delivery of 70 kDa FITC-dextran to primary activated T cells, about 90 minutes after processing, for increasing chip pressures ( n = 2 ).

Article Snippet: For mRNA transfection, we used an mRNA encoding enhanced green fluorescent protein (eGFP) with ARCA cap modifications (Apexbio Technologies, Fisher Scientific #50-199-8310).

Techniques: Expressing, Transfection, Knock-Out