Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) qRT-PCR result of GPR17 mRNA in human gut segments and pancreas (n = 8–10). Gene expression was calculated as 2^-∆∆ct. RPLP0 was used as an internal control. (B) Co-immunostaining of GPR17 and CHGA, a pan-EEC marker, in human small intestine (top) and colon (bottom). (C) Co-immunostaining of GPR17 and GLP-1 in human small intestine (top) and colon (bottom). (D) Co-immunostaining of GPR17 and GIP in human small intestine. (E) Quantification of cells expressing CHGA in GPR17 + cells (n = 117 cells from two human samples were analyzed), and cells expressing GLP-1 in GPR17 + cells (n = 282 cells were analyzed). (F) Quantification of cells expressing GPR17 in GLP-1-producing cells (n = 181 cells from two human samples were analyzed). Data are displayed as means ± SEM. See also .

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Quantitative RT-PCR, Expressing, Immunostaining, Marker

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) (F) FACS of intestine epithelial cells dissociated from Gcg-CreERT;Rosa-tdTomato mouse post tamoxifen injection. Gcg -expressing cells were labeled by red fluorescence (tdTomato) and sorted by FACS within 1 week after tamoxifen treatment (2 mg/d for 5 days by oral gavage). (B–E) qRT-PCR analysis of Gpr17 (B), Gcg (C), Pyy (D), and Gip (E) mRNA in sorted cells (n = 7 mice). Gene expression was calculated as 2 ^ -∆∆ct. β-actin was used as an internal control. Gene expression was expressed relative to input fractions. Unpaired two-tailed Student’s t test, *p < 0.05. Data are displayed as means ± SEM.

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Injection, Expressing, Labeling, Fluorescence, Quantitative RT-PCR, Two Tailed Test

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) Experimental setup for inducible intestinal Gpr17 knockout mice. Gpr17 fl/fl (WT) and Gpr17 fl/fl ;Vil1Cre-ER T2 (iKO) mice were subjected to experiments at least 1 week after tamoxifen treatment (2 mg/d for 5 days by oral gavage). (B) Gpr17 mRNA expression in mouse gut and hypothalamus from iKO (n = 9) and WT (n = 6) mice at 1 week after tamoxifen injection. Gene expression was calculated as 2^-∆∆ct. β-actin was used as an internal control. p values were calculated by unpaired two-tailed Student’s t test. (C–E) Body weight (C), lean mass percent (D), and fat mass percent (E) of male mice before and after tamoxifen injection (n = 8 mice for each group). (F) Fasting body weight before glucose tolerance tests (n = 17~50 mice for each group). (G) Time course study measuring Gpr17 mRNA in duodenum following tamoxifen injection (n = 3 mice). p values were calculated with unpaired two-tailed Student’s t test. (H) Oral glucose tolerance (oGTT, 3 g/kg) in overnight-fasted male iKO (n = 12) and WT (n = 12) mice within 2 months after tamoxifen injection. (I) oGTT (3 g/kg) in overnight-fasted female iKO (n = 14) and WT (n = 12) mice within 2 months after tamoxifen injection. (J) Gpr17 mRNA in duodenum in WT and iKO mice 1 year after initial tamoxifen injection. (K) oGTT (3 g/kg) in 4-h-fasted male iKO (n = 7) and WT (n = 11) mice 1 year after tamoxifen injection. (L) oGTT (3 g/kg) in 4-h-fasted female iKO (n = 10) and WT (n = 6) mice 1 year after tamoxifen injection. Two-way ANOVA and unpaired two-tailed Student’s t test were performed for glucose curve and AUC, respectively. *p < 0.05, **p < 0.01, ***p < 0.001. Data are displayed as means ± SEM. See also .

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Knock-Out, Expressing, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) Oral glucose-stimulated insulin secretion (oGSIS) in overnight-fasted male iKO mice versus WT mice after tamoxifen injection (post-TAM). Serum insulin levels were measured before and 15 min after an oral glucose load (3 g/kg) (n = 10–12 mice). Unpaired two-tailed Student’s t test was performed. (B and C) Plasma total GLP-1 (B) and GIP (C) levels in response to an oral glucose load (3 g/kg) in male inducible intestinal Gpr17 deficient mice (iKO, n = 6) and WT mice (n = 11). Tail blood samples were collected at 0 min and 10 min after oral glucose intake in 4~6 h fasted mice. Two-way ANOVA was performed. (D and E) Plasma total GLP-1 (D) and GIP (E) levels in response to an oral corn oil load (10 µL/g) in male inducible intestinal Gpr17 deficient mice (iKO, n = 6) and WT mice (n = 11). Tail blood samples were collected at 0 min and 60 min after oral glucose intake in 4~6 h fasted mice. Two-way ANOVA was performed. (F) Oral glucose-stimulated insulin secretion (oGSIS) in overnight-fasted female iKO mice versus WT mice after tamoxifen injection (post-TAM). Serum insulin levels were measured before and 15 min after an oral glucose load (3 g/kg) (n = 4~6 mice). Unpaired two-tailed Student’s t test was performed. (G and H) Plasma total GLP-1 (G) and GIP (H) levels in response to an oral glucose load (3 g/kg) in female iKO mice (n = 10) and WT mice (n = 6). Tail blood samples were collected at 0 min and 10 min after oral glucose intake in 4~6 h fasted mice. Two-way ANOVA was performed. (I and J) Plasma total GLP-1 (I) and GIP (J) levels in response to an oral corn oil load (10 µL/g) in female iKO mice (iKO, n = 10) and WT mice (n = 6). Tail blood samples were collected at 0 min and 60 min after oral glucose intake in 4~6 h fasted mice. Two-way ANOVA was performed. (K and L) Gcg (K) and Gip (L) mRNA levels in the gut of inducible Gpr17 deficient mice (iKO) by qRT-PCR (n = 6~9 mice). (M) ipGTT (3 g/kg) in mice pretreated with GLP-1R antagonist exendin-(9–39) (Ex9, 10µg, [I]p.) (n = 3~5 mice). Two-way ANOVA, *p < 0.05 WT + Saline versus iKO + Saline. #p < 0.05, ##p < 0.01 iKO + Saline vs iKO + Ex9. (N) Blood glucose levels at 15 min time point in ipGTT. Unpaired two-tailed Student’s t test was performed. *p < 0.05, **p < 0.01, ***p < 0.001. Data are displayed as means ± SEM.

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Injection, Two Tailed Test, Quantitative RT-PCR

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) Gpr17 mRNA expression in the gut of Gpr17 fl/fl (WT) and Gpr17 fl/fl ;Vil1Cre (KO) mice. Gene expression was calculated as 2^-∆∆ct. β-actin was used as an internal control. Gene expression was expressed relative to duodenum (n = 5 mice for WT, n = 10 mice for KO). Unpaired t tests were performed for statistical analysis. (B) Bright-field images of intestinal organoids derived from adult WT and constitutive Gpr17 KO mice. (C) qRT-PCR of Gpr17 mRNA in organoids (n = 3 mice). Gene expression was normalized to β-actin. Representative gel electrophoresis image of qRT-PCR products was shown. Unpaired two-tailed Student’s t test was performed. (D) qRT-PCR of Gcg and Gip mRNA in organoids (n = 5 mice). (E) GLP-1 (n = 19 replicates for each group) and GIP (n = 7 replicates for each group) secretion from intestinal organoids under basal conditions (1 mmol/L glucose) for 2 h. Unpaired two-tailed Student’s t test was performed. (F) Gpr17 deficiency enhanced fatty acid-induced GLP-1 secretion (n = 7 replicates for each group), but not GIP (n = 5 replicates for each group). Organoids were incubated with palmitoleic acid, a monounsaturated fatty acid, at 40 µmol/L for 2 h. (G) Gpr17 deficiency enhanced bile acid receptor agonist-induced GLP-1 secretion (n = 11 replicates for each group), but not GIP (n = 5 replicates for each group). Organoids were incubated with GPBAR-A, an agonist of bile acid receptor (TGR5), at 20 µmol/L for 2 h. (H and I) Gpr17 deficiency enhanced cAMP-elevating agents-induced GLP-1 secretion (n = 3~13 replicates) (H), but not GIP (n = 3 replicates) (I). Organoids were incubated with cAMP-elevating agents, forskolin (0, 0.3, 1, 3, and 10 µmol/L) and 10 µmol/L IBMX for 2 h. p values were calculated using two-way ANOVA test. Percentage of GLP-1 secretion was calculated by measuring GLP-1 levels in the supernatants and cell lysates and normalized to WT organoids in parallel on the same day. Data were obtained from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. Data are displayed as means ± SEM.

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Two Tailed Test, Incubation

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: (A) Whole-cell patch clamp recording voltage-gated Ca 2+ currents in GLUTag cells that were transiently transfected with pcDNA3.1-mGpr17-GFP. All cells were patched under IR-DIC optics and GFP-positive cells were identified by using an upright microscope fitted with fluorescence optics. (B) Ca 2+ current responses to 150 ms voltage steps of 10 mV increments from a holding potential of −70 mV. The inset shows the voltage pulse protocol. Tetrodotoxin (TTX) at 0.3 µmol/L was added to block Na + currents. (C) Current–voltage relationship of the voltage-gated Ca 2+ currents recorded in Gpr17-overexpressed GLUTag cells (GFP+; n = 27 cells) and nontransfected cells (GFP−; n = 25 cells). Statistical comparisons between groups in were performed using two-way ANOVA and Sidak’s post hoc tests. (D) Averaged AUC for voltage-current curve of Ca 2+ currents. (E) Cell capacitance were comparable between Gpr17-overexpressed GLUTag cells (GFP+) and nontransfected cells (GFP−). (F) Gpr17 expression and agonist stimulation, MDL29,951, inhibited GloSensor cAMP response in GLUTag cells without forskolin addition. (G) Gpr17 expression and MDL29,951 inhibited forskolin-stimulated GloSensor cAMP response in a concentration-dependent manner in GLUTag cells. Data for (F) and (G) were represented as the mean ± SEM of two independent experiments performed with duplicate wells. (H and I) Time course recording showing MDL29,951 inhibited forskolin-stimulated cAMP response in mouse Gpr17-overexpressing GLUTag cells (n = 4 replicates) (H). Peak RLU were quantified (I). Vehicle (Veh) or MDL29,951 (1 µmol/L) were incubated for 10 min before forskolin addition. Unpaired two-tailed Student’s t test was performed. (J) GPR17-Gαi/Gβγ coupling is identified by TRUPATH in HEK293 cells transiently transfected with either pcDNA3.1 (−) empty vector, pcDNA3.1 (+)-hNT1R, or pcDNA3-HA-mGpr17 together with pcDNA5/FRT/TO-Gαi1-Rluc8, pcDNA3.1-Gβ3, and pcDNA3.1-Gγ9-GFP2. The net BRET response represents the BRET ratio for each well subtracted by the mean BRET ratio response of the vehicle-treated wells for each receptor transfection condition. hNT1R, human neurotensin receptor type 1. Data are represented as the mean ± SEM of three or four independent experiments performed with triplicate wells. (K) Schematic diagram illustrates the molecular mechanisms of how GPR17 negatively regulates nutrient-induced GLP-1 secretion in EECs. GPR17 signals through Gαi pathway to reduce cAMP production in enteroendocrine cells, in turn dampening nutrient-stimulated cAMP rise and Ca 2+ -stimulated exocytosis. Gβγ subunits released from Gαi reduce Ca 2+ influx upon nutrient stimulation through activation of G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) channels and inhibition of VDCCs. On the other hand, GPR17 deficiency increases cAMP and intracellular Ca 2+ rise, thereby potentiating nutrient-induced GLP-1 secretion. *p < 0.05, **p < 0.01, ***p < 0.001. Data are displayed as means ± SEM.

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Patch Clamp, Transfection, Microscopy, Fluorescence, Blocking Assay, Expressing, Concentration Assay, Incubation, Two Tailed Test, Plasmid Preparation, Activation Assay, Inhibition

Journal: Cell reports

Article Title: Intestinal Gpr17 deficiency improves glucose metabolism by promoting GLP-1 secretion

doi: 10.1016/j.celrep.2021.110179

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Immunohistochemistry staining of human small intestine (jejunum) and colon were performed by using antibodies for GPR17 (NLS4229; Novus, Centennial, CO), Chromogranin A (SC-393941; Santa Cruz Biotechnology, Dallas, Texas), GIP (ab30679; abcam, Cambridge, MA) and GLP-1 (NBP1–50697; Novus).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 shCON cells were cultured in medium containing 2% FBS, 100% U- 13 C 6 -glucose, and 100% U- 13 C 5 -glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. (A–C) Representative empirical (black) and ISA-derived (red) isotopologue distributions for (A) 16:0, (B) 20:0, and (C) 24:0. The red bracket indicates overestimation of empirical data by ISA modeling, whereas the blue brackets indicate underestimation. (D) ISA sum of squared error (SSE) for the indicated fatty acids normalized to 14:0 ISA. The isotopologue distributions are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. SSEs are the average ± SD of biological quadruplicates. See also .

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Cell Culture, Labeling, Derivative Assay

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 shCON cells were cultured in medium containing 2% FBS, 100% U- 13 C 6 -glucose, and 100% U- 13 C 5 -glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. (A) Schematic of possible contributions to the indicated saturated fatty acids used by FASA. All parameters marked in gray are considered elongated (see for additional details). S , synthesized; I , imported; IE n , imported and elongated n times. (B) Representative empirical (black), ISA-derived (red), and FASA-derived (blue) isotopologue distributions for 16:0. (C) Model-derived contribution parameters for the 16:0 pool. (D) Representative empirical, ISA-derived, and FASA-derived isotopologue distributions for 24:0. The red bracket indicates overestimation of empirical data by ISA modeling, whereas the blue bracket indicates underestimation. (E) Model-derived contribution parameters for the 24:0 pool. (F) SSE for ISA and FASA, normalized to 14:0 ISA. (G) Model-derived D (fractional contribution of 13 C-labeled metabolites to the lipogenic acetyl-CoA) for the indicated fatty acids using ISA or FASA. The isotopologue distributions shown are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. Data shown for pool contribution parameters, SSEs, and D are the average ± SD of biological quadruplicates. See also and .

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Cell Culture, Labeling, Synthesized, Derivative Assay

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 shCON cells were cultured in medium containing 2% FBS, 100% U- 13 C 6 -glucose, and 100% U- 13 C 5 -glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. When applying FASA to polyunsaturated fatty acids (PUFAs), values for D 0 , D 1 , and D 2 were fixed using results from 16:0 to facilitate modeling. (A) Schematic of possible contributions to the indicated n-6 PUFAs used by FASA. All parameters marked in gray are considered elongated. (B) Representative empirical isotopologue distribution and FASA modeling for 22:4n-6. (C) FASA-derived contribution parameters for the 22:4n-6 pool. (D) Representative empirical isotopologue distribution and FASA modeling for 20:3n-6. (E) FASA-derived contribution parameters for the 20:3n-6 pool. The isotopologue distributions shown are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. Data shown for pool contribution parameters are the average ± SD of biological quadruplicates. See also .

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Cell Culture, Labeling, Derivative Assay

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 cells were cultured in a sub-confluent state for 5 days in medium containing 5% FBS and 100% U- 13 C 6 -glucose to achieve iSS and mSS. On day 5, cells were collected, and isotopologue distributions were determined for fatty acids. (A) FASA-derived D parameter values from the indicated SFAs and MUFAs. (B) FASA-derived contribution parameters for selected SFA pools. (C) Diagram for determining elongation and import contribution possibilities to the 20:0 pool. (D) Elongation contribution for the indicated SFAs, MUFAs, and PUFAs. When applying FASA to PUFAs (but not SFAs or MUFAs), values for D 0 , D 1 , and D 2 were fixed using results from 16:0 to facilitate modeling. Data shown are the average ± SD of biological quadruplicates.

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Cell Culture, Derivative Assay

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 shCON cells were treated with 15 ng/mL doxycycline for 96 hr. In the final 48 hr of doxycycline treatment, they were also labeled in medium containing 5% FBS, 100% U- 13 C 6 -glucose, and 100% U- 13 C 5 -glutamine. After 48 hr of labeling, cells were counted on-plate, collected, and analyzed for fatty acid content. When applying FASA, D 0 , D 1 , and D 2 values for all fatty acids were fixed using results from 16:0 to facilitate modeling. (A) Accumulated n-6 PUFA elongation products. (B) Schematic for which SFA and MUFA origins require 18-to-20 elongation; X = 0 or 1 desaturations in fatty acids. (C) Accumulated 20-, 22-, and 24-carbon SFAs and MUFAs that were subject to 18-to-20 elongation during the 48-hr labeling period. (D) Summation of all accumulated 18-to-20 elongation products as described in (C). (E) Accumulated SFA and MUFA elongation products. (F) Accumulated FASN products. For (B)–(D), red, green, and blue indicate contributions from 20-, 22-, and 24-carbon fatty acids, respectively. Values represent average ± SD of biological quadruplicates.

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Labeling

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: H1299 shCON and H1299 shSCAP cells were treated with 15 ng/mL doxycycline for 96 hr. In the final 48 hr of doxycycline treatment, they were also labeled in medium containing 5% FBS, 100% U- 13 C 6 -glucose, and 100% U- 13 C 5 -glutamine. After 48 hr of labeling, cells were collected for gene expression analysis or counted on-plate and collected for fatty acid analysis. When applying FASA, D 0 , D 1 , and D 2 values for all fatty acids were fixed using results from 16:0 to facilitate modeling. (A) mRNA expression of SCAP and selected SREBP targets with doxycycline. (B) Accumulated SFA and MUFA elongation products. (C) Accumulated FASN products. (D) Accumulated n-6 PUFA elongation products. Values represent average ± SD (n = 3 for RNA, n = 4 for fatty acids). ns (not significant) p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed heteroscedastic Student’s t test).

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Labeling, Gene Expression, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

doi: 10.1016/j.celrep.2018.11.041

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: H1299 (human male non-small cell lung cancer-derived; also referred to as NCI-H1299) cells were purchased from ATCC.

Techniques: Virus, Recombinant, Red Blood Cell Lysis, Generated, Staining, Reverse Transcription, Software

Journal: Cell reports

Article Title: Dual roles for piRNAs in promoting and preventing gene silencing in C. elegans

doi: 10.1016/j.celrep.2021.110101

Figure Lengend Snippet:

Article Snippet: NEBNext Small RNA Library Prep Kit for Illumina , New England Biolabs , Cat# E7300S, E7580S.

Techniques: Recombinant, SYBR Green Assay, Expressing, TaqMan microRNA Assay, Sequencing, Software

Journal: Frontiers in Immunology

Article Title: Non-Coding RNAs in Human Breast Milk: A Systematic Review

doi: 10.3389/fimmu.2021.725323

Figure Lengend Snippet: Overview of the methods and aims.

Article Snippet: Rubio et al. ( ) , Agilent RNA 6000 pico chip and Agilent 2100 Bioanalyzer , NEBNext Small RNA Library Prep Set , Illumina HiSeq 2000, single-end 50 bp , QC was conducted using FASTX-Toolkit and FastQ Screen. After adaptor removal, reads with the following features were removed: Reads < 18nt, Mean PHRED scores < 30, and Low complexity reads based on mean score of the read. , • miRBase v21 using miraligner to detect miRNAs and isomiRs • hs37d5 using bowtie and hotspots identified • Hotspots with >60% sequence sharing annotated to different RNA species using miRBase v20, refGene, wgRna, rmsk and tRNAs. , Normalization using DESeq 2 v1.10.1 – internal normalization where geometric mean is calculated for each gene across all samples (scaling factor method). , GSE107524.

Techniques: Isolation, Next-Generation Sequencing, RNA Sequencing Assay, Sequencing, Purification, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Centrifugation, Filtration, In Vitro, Amplification, Microarray, Recombinant, Preserving

Journal: Frontiers in Immunology

Article Title: Non-Coding RNAs in Human Breast Milk: A Systematic Review

doi: 10.3389/fimmu.2021.725323

Figure Lengend Snippet: Methods of quantification in studies using qPCR and miRNA investigated.

Article Snippet: Rubio et al. ( ) , Agilent RNA 6000 pico chip and Agilent 2100 Bioanalyzer , NEBNext Small RNA Library Prep Set , Illumina HiSeq 2000, single-end 50 bp , QC was conducted using FASTX-Toolkit and FastQ Screen. After adaptor removal, reads with the following features were removed: Reads < 18nt, Mean PHRED scores < 30, and Low complexity reads based on mean score of the read. , • miRBase v21 using miraligner to detect miRNAs and isomiRs • hs37d5 using bowtie and hotspots identified • Hotspots with >60% sequence sharing annotated to different RNA species using miRBase v20, refGene, wgRna, rmsk and tRNAs. , Normalization using DESeq 2 v1.10.1 – internal normalization where geometric mean is calculated for each gene across all samples (scaling factor method). , GSE107524.

Techniques: Expressing, SYBR Green Assay, TaqMan Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Non-Coding RNAs in Human Breast Milk: A Systematic Review

doi: 10.3389/fimmu.2021.725323

Figure Lengend Snippet: Methods of quantification in studies using RNA sequencing.

Article Snippet: Rubio et al. ( ) , Agilent RNA 6000 pico chip and Agilent 2100 Bioanalyzer , NEBNext Small RNA Library Prep Set , Illumina HiSeq 2000, single-end 50 bp , QC was conducted using FASTX-Toolkit and FastQ Screen. After adaptor removal, reads with the following features were removed: Reads < 18nt, Mean PHRED scores < 30, and Low complexity reads based on mean score of the read. , • miRBase v21 using miraligner to detect miRNAs and isomiRs • hs37d5 using bowtie and hotspots identified • Hotspots with >60% sequence sharing annotated to different RNA species using miRBase v20, refGene, wgRna, rmsk and tRNAs. , Normalization using DESeq 2 v1.10.1 – internal normalization where geometric mean is calculated for each gene across all samples (scaling factor method). , GSE107524.

Techniques: Sequencing, Expressing, Software, Multiplex Assay, Sample Prep, Transformation Assay