Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: Single-cell gene expression analysis strategy. (A) Experimental design of the single-cell gene expression assay. HSCs were clonally sorted into 96-well PCR plates containing initial reaction mix, including Ambion ® ERCC RNA Spike-In Mixes for quality control. Using the indicated primers, an RT reaction followed by an SMART reaction was used to synthesize first-strand cDNAs with adapter sequences at both the 5′ and 3′ ends. Subsequently, cDNA was amplified by 20 cycles of PCR using a primer containing the adapter sequences, and these amplicons were used as the templates for single-cell real-time RT-PCR. (B) For quality control of these templates, the threshold cycle (Ct) values of control ERCC RNA were examined by real-time PCR simultaneously with the target genes. As the first step of quality control, only single-cell samples that exhibited Ct values within ±2 cycles of the median Ct of each of 3 control ERCC RNAs in each independent experiment were used. In addition, samples that had no detectable signal for any gene were deemed to be empty samples (lacking a cell), attributed to a cell sorting error, and were removed from the analysis. These samples repeatedly showed negative signals for all target genes even when templates were further amplified through 10 additional cycles of PCR (data not shown).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Gene Expression, Control, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, FACS

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: B2m is stably expressed in HSCs, even after ex vivo culture. (A) CD150 + CD34 − KSL cells were cultured for 5 days under 3 different conditions in the presence of SCF and/or TPO. After the culture, CD48 − KSL cells, referred to as the cultured HSC population , were clonally sorted and subsequently subjected to single-cell gene expression analysis. (B) Frequency of cells in which the expression of the indicated genes could be detected before and after culture. The numbers in parentheses represent cells that had positive signals/examined cells under each condition. (C) Heat maps showing the expression values of the indicated genes in individual HSCs before and after the culture under the 3 different conditions. The expression levels of these genes were normalized to the Ct values of ERCC-00130, an exogenously added control RNA. (Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Stable Transfection, Ex Vivo, Cell Culture, Gene Expression, Expressing, Control

Journal: Regenerative Therapy

Article Title: β2-Microglobulin is an appropriate reference gene for RT-PCR-based gene expression analysis of hematopoietic stem cells

doi: 10.1016/j.reth.2015.04.003

Figure Lengend Snippet: The expression level of B2m in HSCs is relatively independent of the influence of ex vivo culture. Using the results of single-cell real-time RT-PCR, changes in the expression levels of the indicated genes in HSCs were examined after ex vivo culture. The expression levels of these genes in individual single-cell samples were normalized to the Ct values of ERCC-00130, an external control RNA. The graphs show relative expression values of Actb (A), B2m (B), Gapdh (C) and Hprt (D). Data are shown as mean ± S.D. (** p < 0.05, Fresh: n = 159, SCF + TPO: n = 148, SCF: n = 163, TPO: n = 127).

Article Snippet: The following pre-made TaqMan ® MGB probes and primer sets were purchased from Life Technologies: beta-actin ( Actb ) (Mm00607939_s1, Mm01205647_g1), B2m (Mm00437762_m1, Mm00437764_m1), glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) (Mm99999915_g1), hypoxanthine phosphoribosyl transferase ( Hprt ) (Mm01318741_m1, Mm00446968_m1), ERCC-00130 (Ac03459943_a1), ERCC-00002 (Ac03459872_a1), ERCC-00113 (Ac03459936_a1).

Techniques: Expressing, Ex Vivo, Quantitative RT-PCR, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene ( nimJ )

doi: 10.1128/AAC.00386-13

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Position Broad Institute locus tag a RAST ID b Recognized by universal primers c Start Stop HMW615 1.1 238 735 HMPREF1204_00002 fig|1073387.3.peg0.9 No HMW615 1.2 1550932 1551429 HMPREF1204_02912 fig|1073387.3.peg.1957 No HMW615 1.5 215 712 HMPREF1204_04081 fig|1073387.3.peg.4096 No HMW616 1.2 1736505 1736008 HMPREF1205_01450 fig|6666666.20506.peg.2695 No HMW610 1.5 1033 458 HMPREF1203_04663 fig|6666666.20571.peg.4745 Yes Open in a separate window a HMPREF1204_00002, HMPREF1204_02912, and HMPREF1204_04081 are annotated in the Broad annotation as Bacteroides fragilis HMW615 hypothetical protein (498 nucleotides [nt]).

Techniques: Plasmid Preparation, Activity Assay, Introduce, Over Expression

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene ( nimJ )

doi: 10.1128/AAC.00386-13

Figure Lengend Snippet: nim genes in B. fragilis HMW610, -615, and -616

Article Snippet: Position Broad Institute locus tag a RAST ID b Recognized by universal primers c Start Stop HMW615 1.1 238 735 HMPREF1204_00002 fig|1073387.3.peg0.9 No HMW615 1.2 1550932 1551429 HMPREF1204_02912 fig|1073387.3.peg.1957 No HMW615 1.5 215 712 HMPREF1204_04081 fig|1073387.3.peg.4096 No HMW616 1.2 1736505 1736008 HMPREF1205_01450 fig|6666666.20506.peg.2695 No HMW610 1.5 1033 458 HMPREF1203_04663 fig|6666666.20571.peg.4745 Yes Open in a separate window a HMPREF1204_00002, HMPREF1204_02912, and HMPREF1204_04081 are annotated in the Broad annotation as Bacteroides fragilis HMW615 hypothetical protein (498 nucleotides [nt]).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene ( nimJ )

doi: 10.1128/AAC.00386-13

Figure Lengend Snippet: (A) Dali Lite superimposed models of NimJ and closest PDB match. Phyre-predicted PDB structure of B. fragilis of NimJ superimposed on the closest match in the PDB database. The highest match was 2furA2 (Z score, 23.3). The NimJ backbone is rainbow colored from the N terminal to the C terminal. (B) Dali pairwise superimposed models: 1W3R.pdb (DR NimA soaked with metronidazole, pyruvate, and acetate) and PDB file of HMW615 NimJ (generated from Phyre server). The yellow band is tyrosine 111. The positions of metronidazole, pyruvate, and acetate molecules are indicated. DR NimA is in the cartoon mode, colored by sequence conservation (identical sequence is in red) with HMW615 NimJ.

Article Snippet: Position Broad Institute locus tag a RAST ID b Recognized by universal primers c Start Stop HMW615 1.1 238 735 HMPREF1204_00002 fig|1073387.3.peg0.9 No HMW615 1.2 1550932 1551429 HMPREF1204_02912 fig|1073387.3.peg.1957 No HMW615 1.5 215 712 HMPREF1204_04081 fig|1073387.3.peg.4096 No HMW616 1.2 1736505 1736008 HMPREF1205_01450 fig|6666666.20506.peg.2695 No HMW610 1.5 1033 458 HMPREF1203_04663 fig|6666666.20571.peg.4745 Yes Open in a separate window a HMPREF1204_00002, HMPREF1204_02912, and HMPREF1204_04081 are annotated in the Broad annotation as Bacteroides fragilis HMW615 hypothetical protein (498 nucleotides [nt]).

Techniques: Generated, Sequencing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene ( nimJ )

doi: 10.1128/AAC.00386-13

Figure Lengend Snippet: Difference in transcription levels of nim genes between clinical isolates and B. fragilis 638R carrying pMCL140:: nim

Article Snippet: Position Broad Institute locus tag a RAST ID b Recognized by universal primers c Start Stop HMW615 1.1 238 735 HMPREF1204_00002 fig|1073387.3.peg0.9 No HMW615 1.2 1550932 1551429 HMPREF1204_02912 fig|1073387.3.peg.1957 No HMW615 1.5 215 712 HMPREF1204_04081 fig|1073387.3.peg.4096 No HMW616 1.2 1736505 1736008 HMPREF1205_01450 fig|6666666.20506.peg.2695 No HMW610 1.5 1033 458 HMPREF1203_04663 fig|6666666.20571.peg.4745 Yes Open in a separate window a HMPREF1204_00002, HMPREF1204_02912, and HMPREF1204_04081 are annotated in the Broad annotation as Bacteroides fragilis HMW615 hypothetical protein (498 nucleotides [nt]).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Two Multidrug-Resistant Clinical Isolates of Bacteroides fragilis Carry a Novel Metronidazole Resistance nim Gene ( nimJ )

doi: 10.1128/AAC.00386-13

Figure Lengend Snippet: MICS of metronidazole for B. fragilis clinical isolates and laboratory constructs

Article Snippet: Position Broad Institute locus tag a RAST ID b Recognized by universal primers c Start Stop HMW615 1.1 238 735 HMPREF1204_00002 fig|1073387.3.peg0.9 No HMW615 1.2 1550932 1551429 HMPREF1204_02912 fig|1073387.3.peg.1957 No HMW615 1.5 215 712 HMPREF1204_04081 fig|1073387.3.peg.4096 No HMW616 1.2 1736505 1736008 HMPREF1205_01450 fig|6666666.20506.peg.2695 No HMW610 1.5 1033 458 HMPREF1203_04663 fig|6666666.20571.peg.4745 Yes Open in a separate window a HMPREF1204_00002, HMPREF1204_02912, and HMPREF1204_04081 are annotated in the Broad annotation as Bacteroides fragilis HMW615 hypothetical protein (498 nucleotides [nt]).

Techniques: