Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A–C) Dose–response curves using double-bubble primer mix #8 and VIC-TqM probe #12 and decimal dilutions of SARS-CoV-2 synthetic RNA N. (A) Real-time amplification plot. C t values: 8 × 10 5 copies, 22.82; 8 × 10 4 copies, 26.27; 8 × 10 3 copies, 30.55; 8 × 10 2 copies, 32.51. (B) 5% agarose gel of the PCR amplification products. Lane 1: ultra-low-range ladder; lanes 2–5: 139-bp amplicons of 8 × 10 5 to 8 × 10 2 RNA copies per tube. Also included in the PCR reaction mix were 2× TqM buffer without UDP and iScript reverse-transcriptase. (C) Efficiency plot of the PCR reaction depicted in panel A. Slope = -3.3359 represents PCR reaction efficiency of 99.42%; R 2 = 0.9816. (D) One-tube RT-qPCR using double-bubble primer mix #10 from region 2 using SARS-CoV-2 synthetic RNA (2 × 10 4 copies per tube) and FAM-TqM probe #13. RT-qPCR was performed with standard conditions.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Amplification, Agarose Gel Electrophoresis, Reverse Transcription, Quantitative RT-PCR

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A) qPCR using reverse-transcribed cDNA template and double-bubble (D-B) primer mix #10, FAM-TqM probe #13, TqM fast kit+ uracil-DNA glycosylase (UDG) assayed in duplicates. Insert depicts 5% agarose gel; lane 1: ultra-low-range (ULR) ladder; lanes 2 & 3: template cDNA; lanes 4 & 5: non-template control (NTC). C t average values: cDNA, 28.60; NTC, undetermined. (B) One-tube RT-qPCR using SARS-CoV-2 synthetic RNA N template and D-B primer mix #8, VIC-TqM probe #12, TqM fast kit without UDG iScript reverse transcriptase, assayed in duplicate. Insert depicts 5% agarose gel; lane 1: ULR ladder; lanes 2 and 3: template RNA; lanes 4 and 5: NTC. C t average values: RNA, 24.63, NTC, undetermined. (C) Duplex qPCR using SARS-CoV-2 synthetic RNA N template and TqM fast kit with UDG. C t values: primer 8, 26.13; primer 10, 32.19; duplex primers 8 + 10, 26.36 and 30.27, respectively; NTC, undetermined. (D) 5% agarose gel of samples shown in panel C. Lane 1: ULR ladder; lane 2: primer D-B mix #8 (amplicon 139 bp); lane 3: D-B primer mix #10 (amplicon 158 bp); lane 4: duplex of both primers #8 and #10 (amplicons 139 and 158 bp). Arrows (C & D) show duplex amplifications in the same tube with D-B primers #8 and #10. PCR was performed using fast conditions with 40 (A, C & D) and 30 (B) cycles.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Agarose Gel Electrophoresis, Control, Quantitative RT-PCR, Amplification

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: The reaction mixture contained hot-start double-bubble (D-B) primer mix #4, VIC-TqM probe #12, cost-effective non-hot-start Taq polymerase (FroggaBio), reverse transcriptase (iScript), synthetic SARS-CoV-2 RNA gene N as template, added dNTPs and ROX dye for internal calibration. The reaction was assembled at room temperature and subjected to real time RT-qPCR using the following fast conditions: 42°C, 15 min; 95°C,1 min; 30 cycles of 95°C, 1 s → 70°C, 20 s. The amplification plot and (insert) 5% agarose gel depict the amplification of SARS-CoV-2 RNA extracted from the nasopharyngeal swabs of patient S1 (lane 2) and negative patient N1 (lane 3), no-template controls (lanes 4 and 5) and SARS-CoV-2 gene N synthetic RNA as positive control (lane 6). Note the amplification in the real-time plot and the 115-bp band in the 5% agarose gel with SARS-CoV-2 virus or synthetic RNA (lanes 2 and 6), with no amplifications in the negative control N1 (lane 3) or NTC (lanes 4 and 5). Lane 1: ultra-low-range ladder. C t values: positive patient S1: 23.95; synthetic RNA: 23.84; negative patient N1 and NTC: undetermined.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Positive Control, Virus, Negative Control

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Frontiers in Pharmacology

Article Title: Establishment of a human nasal epithelium model of histamine-induced inflammation to assess the activity of fexofenadine as an inverse agonist and its link to clinical benefit

doi: 10.3389/fphar.2024.1393702

Figure Lengend Snippet: Reconstituted human nasal epithelium model (MucilAir™) to study the effect of HIS and FEX. CBF, cilia beating frequency; ELISA, enzyme-linked immunosorbent assay; LDH, lactate dehydrogenase; MCC, mucociliary clearance; RT-qPCR, quantitative reverse transcription–polymerase chain reaction; TEER, trans-epithelial electrical resistance.

Article Snippet: The basolateral medium was collected and measured using a Cytotoxicity Lactate Dehydrogenase (LDH) Assay Kit, following the manufacturer’s instructions (WST Dojindo, CK12-20).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction