high capacity cdna reverse transcription kit (Thermo Fisher)

Name:
High-Capacity cDNA Reverse Transcription Kit
Description:
The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single-stranded cDNA in a single 20 µL reaction. The High Capacity cDNA Reverse Transcription Kit delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg total RNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction. Downstream applications include real-time PCR, standard PCR, and microarrays. The kit is ideal for generating cDNA archives.Features of this kit include:• Linear target amplification for real-time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10-fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first-strand synthesis of all RNA species is achieved with the use of the random primers. The kit has been extensively tested with a variety of RNA templates, including GC- and AU-rich targets and RNAs expressed at low levels. cRNA can also be efficiently generated from in vitro transcription of cDNA.
Catalog Number:
4368813
Price:
None
Applications:
PCR & Real-Time PCR|Reverse Transcription
Size:
1 000 reactions
Category:
Kits and Assays, cDNA Synthesis, Library, & Cloning Kits, cDNA Synthesis Kits
Score:
85
|
Buy from Supplier |
Structured Review

The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 µg of total RNA to single-stranded cDNA in a single 20 µL reaction. The High Capacity cDNA Reverse Transcription Kit delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg total RNA. Reactions can be scaled up to 100 µL to generate 10 µg of cDNA from a single reaction. Downstream applications include real-time PCR, standard PCR, and microarrays. The kit is ideal for generating cDNA archives.Features of this kit include:• Linear target amplification for real-time PCR• Higher yields and precision than other cDNA synthesis kits at a fraction of the cost• 10-fold greater dynamic range than other kitsExtensively tested with a variety of templates Quantitative first-strand synthesis of all RNA species is achieved with the use of the random primers. The kit has been extensively tested with a variety of RNA templates, including GC- and AU-rich targets and RNAs expressed at low levels. cRNA can also be efficiently generated from in vitro transcription of cDNA.
https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
Average 99 stars, based on 2431 article reviews
Price from $9.99 to $1999.99
Related Products / Commonly Used Together
Images
1) Product Images from "LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells"
Article Title: LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells
Journal: Oncotarget
doi: 10.18632/oncotarget.21953

Figure Legend Snippet: Reactivation of LINE-1 by BaP is Effected via Canonical TGF-β1 Signaling Total RNA was isolated from BEAS-2B cells treated with 0.5 uM BaP for 8 hours, and 1 µg of RNA subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for ( A ) human LINE-1 (ORF1 and ORF2) or ( B ) TGF-β1. ( C ) cells pre-treated with TGF-β1 receptor inhibitor (LY2157299) or vehicle (DMSO) for 30 min before BaP challenge or ( D ) transfected with target-specific siRNAs to SMAD2, SMAD3, or scramble siRNA or no siRNA (mock) controls. Expression levels are shown as the mean of triplicates with SEM relative to controls. ( E ) Whole cell extracts from transfected cells were analyzed by immunoblotting for SMAD2, SMAD3, SMAD2/3 or GAPDH antibodies (loading control) to confirm target knockdown. Data are representative of two or more independent experiments. Points represent mean of triple samples with SE.
Techniques Used: Isolation, Quantitative RT-PCR, Transfection, Expressing

Figure Legend Snippet: Specificity of TGF-β1/LINE-1 Interactions in Transformed Lung Epithelial Cell Lines NCI-H460, NCI-H520, or NCI-H1993 cell lines were challenged with BaP (0.5–2 uM) or 0.5% DMSO vehicle for 24 hours. Total RNA was isolated and 1 µg subjected to cDNA synthesis. Samples were analyzed by RT-PCR using specific primers for human TGF-β1 ( A ), LINE-1 (L1) ORF 1 ( B ) or GAPDH. Expression levels are presented relative to untreated cells. Each point represents the mean and SE of triplicate samples. The data are representative at two or more independent experiments. ( C ) Cells transfected with target-specific siRNAs to SNAIL or scramble siRNA or no siRNA (mock) were challenged with 3 ng/ml TGF-β1. Whole cell extracts were analyzed by immunoblotting for ORF1p, SNAIL, phospho(p)-SMAD2, or total SMAD2. Data are representative of two or more independent experiments.
Techniques Used: Transformation Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection

Figure Legend Snippet: Activation of EMT Programming by TGF-β1 is Associated with LINE-1 Expression in Human Bronchial Epithelial Cells ( A ) BEAS-2B whole cell lysates isolated from cells stimulated with 3 ng/mL TGF-β1 for 48 hours or control were analyzed by immunoblotting using antibodies against LINE1 (L1) ORF1 protein (ORF1p), E-cadherin, vimentin or GAPDH. ( B ) Total RNA from untreated or treated with 3 ng/ml TGF-β1 for 8 hours, and 1 µg of RNA was subjected to cDNA synthesis. Samples were analyzed by RT-qPCR using specific primers for human L1 (ORF1 and ORF2). Expression levels are shown as the mean of triplicates with SEM relative to controls. ( C ) Whole cell lysates from cells stimulated with different concentrations of TGF-β1 for 48 hours or control were analyzed for expression of L1 ORF1p by immunoblotting. Data are representative of two or more independent experiments.
Techniques Used: Activation Assay, Expressing, Isolation, Quantitative RT-PCR
2) Product Images from "Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells"
Article Title: Anoctamin 1 (Ano1) is required for glucose-induced membrane potential oscillations and insulin secretion by murine β-cells
Journal: Pflugers Archiv
doi: 10.1007/s00424-015-1758-5

Figure Legend Snippet: Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right : molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section . c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm
Techniques Used: Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Positive Control, Negative Control, Sequencing, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Immunohistochemistry, Labeling
3) Product Images from "Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells"
Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells
Journal: Scientific Reports
doi: 10.1038/s41598-018-36312-4

Figure Legend Snippet: LMP1 CTAR1 and CTAR2 contributed to LMP1-mediated increase in sumo levels via the activation of NF-κB. ( a , b ) 293 cells were transfected with 1 μg of a LMP1-expression plasmid, select LMP1 mutant-expression plasmid, or a pcDNA3 control plasmid. ( a ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 and sumo-2/3 levels (relative to gapdh ), The fold change in sumo- 1 and sumo-2/3 levels (compared to control-expressing cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( b ) Slot blots were performed to detect SUMO-1 and SUMO-2/3 levels. Actin was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( c , d ) EBV-transformed LCLs were treated with Bay 11–7082 (1 μM), LY294002 (5 μM), or DMSO (the vehicle control; Control) for 24 hours. ( c ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 , and sumo-2/3 expression (relative to gapdh ), and the fold change in sumo-1 and sumo-2/3 levels (relative to control-treated cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( d ) Slot immunoblots and Western blots analyses were performed to detect SUMO-1 and SUMO-2/3 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( e ) cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells that were treated with DMSO (the vehicle control; control) or Bay 11–7085 (1 μM) for 24 hours. Real-time PCR was performed was performed to quantitate relative sumo-1 , and sumo-2/3 levels (relative to gapdh ). The fold change in sumo-1 and sumo-2/3 levels (relative to control-treated BL41 EBV WT cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate.
Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation, Transformation Assay, Western Blot, Generated

Figure Legend Snippet: Increased sumo-1/2/3 levels were detected in LMP1-positive lymphoma tissues in an LMP1-dependent manner. ( a , b ) RNA was extracted from 42 biopsy tissues and cDNA was made. Real-time PCR was performed to quantitate gapdh , LMP1, and sumo-1 levels. RNA yields allowed sumo-2/3 levels to be tested in 17 of the 42 samples. ( a ) Relative LMP1, sumo-1 and sumo-2/3 levels (relative to gapdh ) were determined. The detection of LMP1 RNA levels determined is tissues were LMP1-negative (LMP1-neg) and LMP1-positive (LMP1-pos). Results are shown as the mean relative sumo-1 and sumo-2/3 levels for individual samples (shapes). Horizontal lines represent man sumo-1 and sumo-2/2 levels for the collective LMP1-neg and LMP1-pos samples. ( b ) Of the 19 LMP1-positive tissue samples, relative sumo- 1 levels were determined in all 19 samples but relative sumo-2/ 3 levels were determined in only 9 samples. Results are shown as the mean relative LMP1, sumo- 1, and sumo-2/ 3 leves of samples run in duplicate. Regression analysis was performed.
Techniques Used: Real-time Polymerase Chain Reaction

Figure Legend Snippet: LMP1 was necessary and sufficient to increase sumo-1/2/3 and SUMO-1/2/3 levels in vivo . ( a ) Real-time PCR was performed using cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells. The fold change in relative sumo- 1 and sumo- 2/3 expression (relative to gapdh ) was determined. Results are shown as the mean ± the standard deviation samples run in triplicate and independent experiments performed in triplicate. ( b – c ) 293 cells were serum-starved for 24 hours and then transfected with graduated amounts of an LMP1-expression vector or a control vector in serum-free media. ( b ) RNA was harvested, cDNA prepared, and real-time PCR performed. Relative LMP1, sumo-1 , and sumo-2/3 levels were determined (relative to gapdh ). Regression analysis of LMP1 and sumo-1 or sumo-2/3 levels was performed. Samples were run in triplicate and independent experiments performed in triplicate. ( c ) Slot blots were performed to detect SUMO-1, SUMO-2/3, and LMP1 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown.
Techniques Used: In Vivo, Real-time Polymerase Chain Reaction, Generated, Expressing, Standard Deviation, Transfection, Plasmid Preparation
4) Product Images from "Promoter of CaZF, a Chickpea Gene That Positively Regulates Growth and Stress Tolerance, Is Activated by an AP2-Family Transcription Factor CAP2"
Article Title: Promoter of CaZF, a Chickpea Gene That Positively Regulates Growth and Stress Tolerance, Is Activated by an AP2-Family Transcription Factor CAP2
Journal: PLoS ONE
doi: 10.1371/journal.pone.0056737

Figure Legend Snippet: Activation of CaZF -promoter by CAP2 in plant cell. (A, B) Enhancement of CaZF expression by transient overexpression of CAP2 in chickpea. Young leaves of 10-day-old chickpea seedlings were transformed with CaMV35S:c-Myc-CAP2 by particle bombardment. Chickpea leaves were harvested after 48 h of incubation. Leaves transformed with empty vector (pCAMBIA1302) were taken as control (Con). 2 µg of total RNA was reverse transcribed for cDNA preparation. CAP2 (A) and CaZF (B) expression was analyzed in the control and experimental tissues by semi-quantitative RT-PCR (27 cycles) and qReal-Time PCR. The expression level of Actin gene was taken as an internal control. Results from two biological replicates (Expt-1, Expt-2) are shown. (C) Schematic diagram of the effector and reporter constructs used in the co-transfection experiments. Full-length CAP2 cDNA was fused with 2X c-Myc at N-terminus and cloned under CaMV-35S promoter in pCAMBIA1302 to construct effector plasmid. pro CaZF with three CRTs was fused with GUS gene in pBI101 to construct reporter plasmid. (D) Both the effector and reporter plasmids as mentioned in the figure were co-introduced in to tobacco leaf explants by Agrobacterium -mediated transformation and antibiotic-selected shootlets were used for the GUS assay. Expression of kanamycin resistance gene (NPT II) as assessed by qRT-PCR was used for normalization of results in the transformed shoot-lets. (E) The effector and reporter constructs were co-introduced into tobacco BY2 protoplasts as mentioned in the table. CAP2 stands for the effector plasmid and pro- CaZF stands for the reporter plasmid. pro- CaZF (M1–M3) stands for the reporter plasmids with mutations in CRT1-CRT3 in pro- CaZF . GUS activity was measured fluorometrically after 48 h of transformation. The empty vectors without CAP2 (pCAMBIA1302) or pro CaZF (pBI101) were used as controls. Transfection efficiency of the CaMV-35S-EYFP1 plasmid included in protoplast experiment was used for normalization. The error bars indicate the standard deviation (SD). * indicates significant differences in comparison to the controls at p
Techniques Used: Activation Assay, Expressing, Over Expression, Transformation Assay, Incubation, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction, Construct, Cotransfection, Clone Assay, GUS Gene Assay, Activity Assay, Transfection, Standard Deviation

Figure Legend Snippet: Expression analysis of CaZF in response to various stresses in chickpea. (A) Expression patterns of CaZF gene induced under different treatments. Total RNA was isolated from 10-d-old chickpea seedling treated with distilled water (control, Con), 100 µM abscisic acid (ABA), 4°C (Cold), dehydration (DH), 50 µM methyl-jasmonate (MeJA), 5 mM salicylic acid (SA), 250 mM NaCl (Salt) and wound for 5 h. (B) Time-course of accumulation of CaZF mRNA under dehydration, cold and salt treatments for 0.5 h, 1 h and 3 h. (C) Organ-specific expression of CaZF in root (R), shoot (S) and leaf (L) after cold, salt and dehydration treatments for 3 h. 20 µg of total RNA was electrophoresed on formaldehyde denaturing gel, blotted onto nylon membrane and hybridized with 32 P-radiolabeled CaZF cDNA. The lower panel in each figure shows ethidium bromide-stained ribosomal RNAs as loading controls. The relative intensities of the bands were quantitated by densitometry in PhosphorImager scanner and are presented in the form of fold changes below each blot.
Techniques Used: Expressing, Isolation, Staining
5) Product Images from "Modeling Acute ER stress in vivo and in vitro"
Article Title: Modeling Acute ER stress in vivo and in vitro
Journal:
doi: 10.1097/SHK.0000000000000759

Figure Legend Snippet: Tunicamycin is a more robust inducer of ER stress in Adipocytes A) Oil Red O staining confirming full differentiation of 3T3-L1 cells to mature adipocytes before treatments were initiated. B-E) 3T3-L1 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. F) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TUN (5ug) for 24hrs. Thapsigargin (TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TG. (n=6).
Techniques Used: Staining, Real-time Polymerase Chain Reaction, Immunostaining

Figure Legend Snippet: Tunicamycin and thapsigargin are equally induce ER stress in Hepatocytes A-D) HepG2 cells were exposed to either TG (25nM, 50nM, 100nM) or TUN (2.5ug, 5ug, 10ug) for 24 Hrs, and total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated ER stress/UPR genes were quantified using real-time PCR and normalized to 18S rRNA. E) Immunostaining of cleaved-ATF6 (red), ER (green), and nuclei (blue) upon stimulation with vehicle or TG (100 mM) for 24hrs. F) Cell viability assay were performed after HepG2s were incubated with various doses of TG or TUN based on quantitation of the ATP present. Thapsigargin TG); tunicamycin (Tun). Data shown are in mean ± SEM, *p < 0.05 vs vehicle, # p < 0.05 vs TUN (5ug) (n=6).
Techniques Used: Real-time Polymerase Chain Reaction, Immunostaining, Viability Assay, Incubation, Quantitation Assay
6) Product Images from "Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development"
Article Title: Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development
Journal: International Journal of Molecular Sciences
doi: 10.3390/ijms16034492

Figure Legend Snippet: Characterization of primer pairs designed for expression analysis of the internal control during mouse lung development. ( A ) Amplification efficiency for each primer pair was calculated using 10-fold serial dilutions of a cDNA template. Primer amplification efficiency was assessed by plotting the cycle threshold ( C T ) value for each concentration against the logarithm (base 10) of the fold dilution (log 10 (Quantity)). Efficiency was calculated using the slope of the linear function; ( B ) Dissociation curve analysis of primer specific products was performed by constantly monitoring the fluorescence with increasing temperatures from 60 to 95 °C. Melt curves were generated by plotting the negative first derivative of the fluorescence (−d/d T (Fluorescence) 520 nm) versus temperature (degree Celsius, °C); ( C ) Agarose gel electrophoresis after qRT-PCR indicates a specific product for each of the primer pairs. +RT, template is cDNA; −RT, template is RNA without reverse transcriptase, W, water control.
Techniques Used: Expressing, Amplification, Concentration Assay, Fluorescence, Generated, Agarose Gel Electrophoresis, Electrophoresis, Quantitative RT-PCR
7) Product Images from "Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury"
Article Title: Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury. Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury
Journal: Physiological Reports
doi: 10.14814/phy2.13753

Figure Legend Snippet: Compound 1 treatment of human lung fibroblasts cell cultures increases intracellular cAMP and decreases TGF ‐ β ‐mediated induction of fibrotic gene expression. WI ‐38 human lung fibroblasts were seeded on 24‐well plates and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L). After 30 min, cells were stimulated by 1 μ mol/L forskolin for 30 min and cell lysates were analyzed for cAMP concentration (A). To analyze fibroblast gene expression, WI ‐38 human lung fibroblasts were cultured in media containing 0.5% FBS for 24 h and treated with increasing concentrations of Compound 1 (1 × 10 −10 mol/L to 1 × 10 −5 mol/L) for 1 h followed by TGF ‐ β (3 ng/mL) and forskolin (1 μ mol/L) for 24 h. Total RNA was extracted from cell lysates, cDNA was amplified, and target gene mRNA was measured. The target gene expression levels of Col1a1 (Type‐1 collagen), Fn (Fibronectin), CTGF (connective tissue growth factor), and PAI ‐1 (plasminogen activator inhibitor‐1) was normalized to Glyceraldehyde 3‐phosphate dehydrogenase ( GAPDH ). Data are reported as mean ± SD with individual data points shown ( n = 3 per group). # P ≤ 0.05 versus forskolin group by two‐tailed Williams’ test.
Techniques Used: Expressing, Concentration Assay, Cell Culture, Amplification, Two Tailed Test

Figure Legend Snippet: Therapeutic administration of Compound 1 to targeted type II AEC ‐injured mice reduces the expression of TNF α within the lung. DTR ‐expressing mice ( DTR +) were administered daily I.P. PBS or DT from day 0 through Day 14. Subsets of the DTR +: DT ‐treated animals were treated by oral gavage once daily beginning on day 11 with vehicle or Compound 1 at 5.0 mg/kg. On day 21, the left lung was harvested and homogenized, and total RNA was extracted. First‐strand cDNA was synthesized and mRNA levels for Col1a1, Fibronectin, CTGF TNF α and PAI ‐1 (plasminogen activator inhibitor‐1) were assessed using SYBR Green‐based detection. The expression levels were normalized to GAPDH using the following formula: % GAPDH expression = 100/2‐ΔΔ CT . Data are presented as an average ± SEM ( n = 9 per DTR +: DT vehicle‐ and drug‐treated groups).
Techniques Used: Mouse Assay, Expressing, Synthesized, SYBR Green Assay
8) Product Images from "Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis"
Article Title: Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis
Journal: PLoS ONE
doi: 10.1371/journal.pone.0115099

Figure Legend Snippet: Spo11 is spliced less in asexual aphids than in sexual aphids. A. Pea aphid Spo11 gene structure is shown. Boxes refer to exons and lines refer to introns, with bp lengths indicated inside or below, respectively. The third intron includes a 1720 bp SINE2 retrotransposon, indicated by the arrowhead. PCR primer sets used in reactions shown in Panels B , C and D are indicated by a numbered dashed line and pair of arrows. The scale bar indicates 100 bp. B. Unspliced Spo11 is detected in asexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from sexual (“Sex”) LSR1.G1.AC ovaries, asexual (“Asex”) LSR1.G1.AC ovaries (+) and Tucson (“TUC”) pea aphid ovaries; and the RT-absent control template (-). Upper panel shows PCRs products from the second and third exons of Spo11 . The bottom panel shows PCR products of the last four ( left) or last six ( right ) exons of Spo11 . DNA size standards are indicated on the marker (M) lanes of these gels. C. Spo11 intron RNA is more abundant in asexually reproducing aphids than in sexual aphids. Shown are PCRs from: genomic DNA (g); cDNA pools synthesized from RNA from Tucson ovaries, asexual or sexual LSR1.G1.AC ovaries (+); or the RT-absent control template (-). Upper panel shows PCRs products from the fourth intron of Spo11 . The bottom panel shows PCR products of full-length Hop2 using the same template amounts. D. Spo11 is expressed in asexual embryos in utero . Top panel shows Spo11 PCR products using primer set 2, and the bottom panel shows Pms1 PCR product. Pools of cDNA (+) or control template from cDNA synthesis reactions lacking RT (-) were created from the tissue sources listed above the lanes.
Techniques Used: Polymerase Chain Reaction, Synthesized, Marker, In Utero

Figure Legend Snippet: Argonaute -family genes are differentially expressed between aphid morphs. Shown are PCRs of aphid Argonaute -family genes Piwi3 , Piwi8 and Ago3a , along with GAPDH as a loading control. Products were amplified from cDNA pools synthesized with (+) or without (-) RT from RNA from asexual or sexual LSR1.G1.AC ovaries.
Techniques Used: Amplification, Synthesized
9) Product Images from "A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype"
Article Title: A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype
Journal: Cancer Growth and Metastasis
doi: 10.4137/CGM.S18581

Figure Legend Snippet: Overexpression of recombinant cavin-3 specifically decreases cell migration and abrogates PMA-induced MMP-9 expression. ( A ) Human HT-1080 fibrosarcoma cells were transiently transfected with 2 μg of cDNA plasmids and fluorescent confocal microscopy was used to visualize the encoded recombinant GFP-tagged cavin-1, cavin-2 or cavin-3 proteins 24–48 hours post transfection. pEGFP cDNA plasmid was used as control. ( B ) Cells were then harvested and cell migration assayed as described in the Methods section. ( C ) HT-1080 cells were transiently transfected with plasmids as described above. Twenty-four hours post transfection, cells were treated with vehicle (white bars) or 100 nM PMA (Black bars) in a serum-free medium for 18 hours. Total RNA was extracted and qRT-PCR was performed to measure the levels of MMP-9 transcript. Values of PCR are normalized over the expression of housekeeping genes GAPDH and PPI γ and are the mean ± S.E.M of triplicate values from one representative experiment.
Techniques Used: Over Expression, Recombinant, Migration, Expressing, Transfection, Confocal Microscopy, Plasmid Preparation, Quantitative RT-PCR, Polymerase Chain Reaction
10) Product Images from "Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells"
Article Title: Tetracycline Derivative Minocycline Inhibits Autophagy and Inflammation in Concanavalin-A-Activated Human Hepatoma Cells
Journal: Gene Regulation and Systems Biology
doi: 10.4137/GRSB.S13946

Figure Legend Snippet: MT1-MMP is required for ConA-induced BNIP3 expression and formation of acidic vacuoles. Serum-starved HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against MT1-MMP (siMT1-MMP); then the cells were treated with 30 μg/mL ConA for 24 hours in the presence or absence of 10 μM minocycline. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods section. ( B ) Total RNA was isolated from cells transfected with either a scrambled siRNA sequence (siScr) or a specific siRNA against MT1-MMP (siMT1-MMP), and followed by 30 μg/mL ConA treatment. cDNA was then synthesized and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance. ( C ) Cells were stained with Acridine Orange as described in the Methods section, and acidic vacuole formation was examined by fluorescence microscopy in control, ConA-, and ConA/minocycline-treated cells.
Techniques Used: Expressing, Transfection, Isolation, Immunodetection, Sequencing, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Microscopy

Figure Legend Snippet: STAT3 and NANOS1 gene silencing abrogate ConA-induced MT1-MMP and BNIP3 expression. HepG2 hepatoma cells were transiently transfected with a control siRNA (siScrambled) or an siRNA directed against STAT3 (siSTAT3) or NANOS1 (siNANOS1); then serum-starved cells were treated with 30 μg/mL ConA for 24 hours. ( A ) Cell lysates were isolated and electrophoresed, and immunodetection performed as described in the Methods sections. ( B ) MT1-MMP gene expression was assessed in cells that were treated with 10 μM minocycline or 30 μg/mL ConA. Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. ( C ) The inter-relationship between NANOS1 and STAT3 gene expression was assessed in cells that were either silenced for NANOS1 (siNANOS1, black bars) or silenced for STAT3 (siSTAT3, gray bars). Total RNA was isolated, cDNA synthesized, and qRT-PCR performed as described in the Methods section. A representative qPCR profile, out of three independent experiments, is shown for the corresponding genes. Data represent mean values in triplicates. Statistical significance was assessed using Student’s unpaired t -test. Probability values of less than 0.05 were considered significant, and an asterisk (*) denotes such significance.
Techniques Used: Expressing, Transfection, Isolation, Immunodetection, Synthesized, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Figure Legend Snippet: Minocycline inhibits ConA-induced NANOS1 expression and phosphorylation of STAT3. ( A ) Serum-starved HepG2 hepatoma cells were treated with increasing ConA concentrations. NANOS1 and MT1-MMP gene expression was expressed as the percentage of maximal ConA-induced effect for each gene. ( B ) ConA-treated HepG2 hepatoma cells were incubated with up to 10 μM minocycline for 24 hours. Total RNA was isolated, cDNA synthesized, and qPCR performed as described in the Methods section. The levels of STAT3 phosphorylation were assessed in cells that were ( C ) treated with 30 μg/mL ConA in the presence or absence of 10 μM minocycline or ( D ) transiently transfected with either a control siRNA (siScrambled) or an siRNA directed against NANOS1 (siNANOS1). A representative qPCR profile, from two independent experiments, is shown for the corresponding genes. Data represent mean values from triplicates.
Techniques Used: Expressing, Incubation, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Transfection
11) Product Images from "Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime"
Article Title: Correction of RT-qPCR data for genomic DNA-derived signals with ValidPrime
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkr1259
![... assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in ... Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with](https://storage.googleapis.com/bioz_article_images/PMC3326333/gkr1259m7.jpg)
Figure Legend Snippet: Correction of endogenous gDNA with ValidPrime. Comparison of results obtained with two assays targeting Serpine1 (left) or Chi3l3 (right) in cDNA prepared from mouse peritoneal macrophages and measured in the BioMark qPCR system. The ‘gDNA-sensitive’ assays amplify both gDNA and cDNA, while the ‘gDNA-insensitive’ assays only recognize the transcript. ( A ) Scatter plots showing the correlation between the %DNA [as defined in Equation (6) ] and Cq NA data obtained with the gDNA-sensitive assays in each of 81 independent RNA preparations (means of duplicates). The positive correlation between %DNA and Cq illustrates the increasing impact of the gDNA contamination with decreasing total signal. Mean and median values refer to %DNA levels. ( B ) Cq NA data measured with the gDNA-insensitive assays plotted against the corresponding Cq NA data (dark blue) or ValidPrime-estimated Cq RNA (light blue and orange), obtained with the gDNA-sensitive assays. Samples with a DNA contribution of 60–90 % are shown in orange and those with
Techniques Used: Real-time Polymerase Chain Reaction
![... melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ... ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.](https://storage.googleapis.com/bioz_article_images/PMC3326333/gkr1259f5.jpg)
Figure Legend Snippet: ValidPrime flowchart. ValidPrime GOI assay validation. ValidPrime can be used as a reliable, cost-efficient alternative to RT(−) controls to survey gDNA background in RT–qPCR, and as a tool to determine the RNA-derived signal ( Cq RNA ) in RT(+)–qPCR reactions. To optimize its accuracy when Cq RNA calculation is desired, validation of GOI assays in gDNA samples is recommended, as outlined in ( A ). Asterisks indicates the efficiency evaluation and melting curve/electrophoresis-based analysis. This includes an evaluation of the gDNA sensitivity of GOI assays using dilution series with gDNA samples spanning at least three log 10 in copy number. GOI assays that do not amplify gDNA are attributed the grade A+. The amplification of gDNA by high-confidence assays should be specific and with an efficiency similar to that of the VPA (see ‘Discussion’ section and Supplementary Figure S3 ). For GOI assays with suboptimal, but confidently determined ( 17 , 21 ) efficiency, Equation (7) could be applied to adjust Cq NA data. To optimize specificity, there should also be consistency between the melting curves of PCR products in gDNA and cDNA samples. ( B ) Cq RNA calculation with ValidPrime-validated GOI assays. High confidence and A+ assays can be used with less gDNA samples for Cq RNA determination. It is recommended to confirm the absence of gDNA amplification at least once for A+ assays. Samples that do not contain sufficient gDNA to generate a signal with the VPA are attributed A*. As for gDNA insensitive A+ assays, Cq RNA equals Cq NA (i.e. output = input) in A* samples, since the DNA-derived signal is negligible [see Equations ( 2 and 4 )]. For gDNA-sensitive GOI assays, Cq RNA is calculated by a Cq DNA -based correction of Cq NA using Equations ( 4 and 5 ). To minimize the risk of jeopardizing the accuracy of the Cq RNA estimation, it is not advisable to perform correction on samples where the DNA-derived signal exceeds 60%. The calculations are facilitated using the ValidPrime software. Details on additional assay/sample grading and data output formats employed by the software are provided in Supplementary Figure S7 . The Cq RNA output data can be used for downstream data processing, such as normalization against reference genes.
Techniques Used: Quantitative RT-PCR, Derivative Assay, Electrophoresis, Amplification, Polymerase Chain Reaction, Software
12) Product Images from "Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure"
Article Title: Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure
Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
doi: 10.1161/JAHA.114.001104

Figure Legend Snippet: Thapsigargin (TG)‐induced ER stress causes and time‐dependent corin mRNA decay in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free DMEM‐M199 (4:1) with and without 0.1 μmol/L TG for 6, 16, 24, and 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and gene expression of the following genes was assessed by RT‐PCR (A through F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; and (F) ATF4. Compared with non‐TG‐treated controls, cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4, and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P
Techniques Used: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

Figure Legend Snippet: Thapsigargin (TG)‐induced ER stress and time‐dependent corin mRNA decay in adult mouse atrial cardiomyocytes (HL‐1) cells. HL‐1 cells were plated in 6‐well plates. Twenty‐four hours later, cells were cultured in serum‐free Claycomb medium with and without thapsigargin (TG) 0.1 μmol/L for 6, 16, 24, 30 hours, separately. Total RNA was extracted and reverse‐transcribed into cDNA and mRNA abundance of the following genes was assessed by RT‐PCR ( Figure 4 A through 4 F): (A) GRP78; (B) XBP1 splicing; (C) CHOP; (D) corin; (E) GRP94; (F) ATF4. Compared with non‐TG‐treated HL‐1 control cells, HL‐1 cells treated with TG demonstrated increased ER stress as evidenced by significant increases in GRP78, GRP94, ATF4 and CHOP mRNA, with a corresponding significant increase in XBP1 splicing based on the increase in the spliced form (intron removed) XBP1s mRNA; * P
Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction
13) Product Images from "Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1"
Article Title: Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1
Journal:
doi: 10.1128/IAI.01341-09

Figure Legend Snippet: COX-2 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA
Techniques Used: Expressing, Isolation

Figure Legend Snippet: Egr-1 mRNA expression by macrophage-like THP-1 cells. Cells were stimulated with Stx1 (400 ng/ml) (A), LPS (200 ng/ml) (B), or Stx1+LPS (400 and 200 ng/ml, respectively) (C) for different times. Total RNA was isolated, DNase treated, and cDNA
Techniques Used: Expressing, Isolation
14) Product Images from "Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1"
Article Title: Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1
Journal: Life sciences
doi: 10.1016/j.lfs.2014.03.028

Figure Legend Snippet: Time-dependent regulation of ET A and ET B mRNA expressions in the renal inner medulla and cortex. Wild type (light bars) or Per1 het (dark bars) mice were euthanized at noon or midnight. Total RNA was isolated from dissected inner medulla and cortex and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate mRNA expressions of ET A (Panel A) and ET B (Panel B). Data are presented relative to the WT at noon, ±SEM. †P
Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Figure Legend Snippet: Regulation of ET-1, ET A and ET B mRNA expression in the heart by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the heart and converted to cDNA. Real time quantitative RT-PCR (qPCR)was performed to evaluate in ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Figure Legend Snippet: Regulation of ET-1, ET A , and ET B mRNA expressions in the liver by time and Per1. Wild type or Per1 hetmice were euthanized at noon or midnight. Total RNA was isolated from the liver and converted to cDNA. Real time quantitative RT-PCR (QPCR) was performed to evaluate expression of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
Techniques Used: Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing

Figure Legend Snippet: Regulation of the endothelin axis in the lung by time and Per1. Wild type or Per1 het mice were euthanized at noon or midnight. Total RNA was isolated from the lung and converted to cDNA. Real time quantitative RT-PCR (qPCR) was performed to evaluate expressions of ET-1 (Panel A), ET A (Panel B), and ET B (Panel C). Data are presented relative to the WT at noon, ±SEM. *P
Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction
15) Product Images from "Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin"
Article Title: Branched Chain Amino Acid Suppresses Hepatocellular Cancer Stem Cells through the Activation of Mammalian Target of Rapamycin
Journal: PLoS ONE
doi: 10.1371/journal.pone.0082346

Figure Legend Snippet: Changes in the percentage of EpCAM-positive cells upon control medium (DMEM containing 10% FBS) with 4 mM BCAA stimulation or 100 nM rapamycin pretreatment and 4 mM BCAA stimulation (A) or liver cirrhosis modified medium (LC) containing 10% FBS stimulation (B) for 72 h in Huh7 by using the target activation protocol of array scan. The rate change of EpCAM-positive cells in 5000 cells with overexpression of caRheb or control plasmid cDNA (pc DNA) in control medium (DMEM containing 10% FBS) with and without 4 mM BCAA stimulation for 24 h in Huh7 (C). The detection of P70S6 kinase phosphorylation, a member of downstream mTOR signaling, in the presence of DMEM, BCAA treatment, pretreatment with rapamycin and BCAA treatment, or LC stimulation for 72 h in Huh7 (A,B). Tukey’s test **p
Techniques Used: Modification, Liquid Chromatography, Activation Assay, Over Expression, Plasmid Preparation

Figure Legend Snippet: Protein expression and phosphorylation in Huh7 cells under Knockdown conditions for 5 days and overexpression conditions for 1 day, and detection of phosphorylation after 4 mM BCAA treatment for 30 min. Rictor or Raptor Knockdown compared to negative control (NC), caRheb compared to control plasmid cDNA (pc DNA), BCAA treatment compared to DMEM (FBS 10%) only (Ctrl) (A). The average tumor volumes and tumorigenesis ratio at the 4 th week in a xenograft model with transplanted cells with negative control, knockdown of Raptor, Rictor for 5 days, or overexpression of control plasmid DNA, caRheb for 1 day (C), and tumorigenesis rate (B). Dunnett's test, *p
Techniques Used: Expressing, Over Expression, Negative Control, Plasmid Preparation
16) Product Images from "Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells"
Article Title: Na+/K+ ATPase activity promotes invasion of endocrine resistant breast cancer cells
Journal: PLoS ONE
doi: 10.1371/journal.pone.0193779

Figure Legend Snippet: NKP expression and activity in normal and breast cancer cell lines. Panel A: NKP mRNA expression in ER+ (YS 1.2) and ER-ve (pII and MDA-MB-231) breast cancer cells. RNA was extracted from cells, converted to cDNA and PCR amplified. Ct values were converted to ratios relative to actin levels as described in Methods. Panel B: NKP protein expression level in the membranous compartment of the tested cell lines was determined by western blotting. Membranous fraction lysate protein (3 μg) was electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to T-NKP and actin (loading control). This blot represents one of 3 independent determinations. NKP activity in the membranous fraction of all cell lines (panel C), and in pII cells in response to ouabain/TTX treatment (panel D) was determined by a colorimetric NKP activity kit. Histobars represent means ± SEM of at least 3 independent determinations. *denotes significant difference from MCF10A (panel C) or pII control (panel D) with p
Techniques Used: Expressing, Activity Assay, Multiple Displacement Amplification, Polymerase Chain Reaction, Amplification, Western Blot

Figure Legend Snippet: Effect of siRNA-mediated knockdown of NKP on pII cell motility. Panel A: NKP mRNA expression in pII cells transfected with a scrambled sequence (control, open bar) or NKP siRNA (48 h, solid bar). RNA was extracted from cells, converted to cDNA and amplified by PCR. Ct values were converted to ratios relative to actin. Panel B: mean distance moved (in pixels) as a measurement of cell motility for pII cell transfected with a scrambled sequence (control, open bar, taken as 100%) or NKP siRNA (48 h, solid bar) determined after 24 h of incubation. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from control with p
Techniques Used: Expressing, Transfection, Sequencing, Amplification, Polymerase Chain Reaction, Incubation
17) Product Images from "HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells"
Article Title: HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells, et al. HNRNPLL stabilizes mRNA for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells
Journal: Cancer Science
doi: 10.1111/cas.13660

Figure Legend Snippet: HNRNPLL promotes cell cycle progression in colon cancer cells. A‐D, Cell cycle analysis for SW 480 (A) and HT 29 (C) cells transduced with HNRNPLL cDNA , Luc sh RNA or HNRNPLL sh RNA 2 was performed using a flow cytometer. The sum of the percentages of S and G2/M phases in SW 480 (B) and HT 29 (D) cells are shown in the bar graph. Error bars, SD . E, Immunostaining of HT 29 cells using antibodies for HNRNPLL , GMNN and CDT 1. Note that cells showing no nuclear GMNN expression and high CDT 1 expression (*) exhibit higher HNRNPLL expression compared to cells showing nuclear GMNN expression and low CDT 1 expression ( † ). Scale bar, 10 μm
Techniques Used: Cell Cycle Assay, Transduction, Flow Cytometry, Cytometry, Immunostaining, Expressing

Figure Legend Snippet: Knockdown of PCNA , RFC 3 or FEN 1 suppresses the increased cell proliferation caused by HNRNPLL overexpression. A, Western blot analysis to confirm the overexpression of HNRNPLL and knockdown of PCNA , RFC 3 and FEN 1. Arrows and arrowheads indicate FLAG ‐tagged or endogenous HNRNPLL , respectively. B, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced SW 480 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .036 (*), .20 ( † ), .32 ( †† ), .12 ( ††† ), .030 ( ‡ ), .038 ( ‡‡ ), .027 ( ‡‡‡ ), .50 ( § ), .78 ( §§ ) and .21 ( §§§ ). C, MTT assay was performed for Mock‐ or HNRNPLL cDNA ‐transduced HT 29 cells 72 h after transfection of control si RNA or si RNA (s) targeting PCNA , RFC 3 , or FEN 1 . Error bars, SD . P = .026 (*), .079 ( † ), .79 ( †† ), .12 ( ††† ), .036 ( ‡ ), .040 ( ‡‡ ), .033 ( ‡‡‡ ), .12 ( § ), .34 ( §§ ) and .18 ( §§§ ). D, Western blot analysis to confirm the overexpression of FLAG ‐ PCNA , FLAG ‐ RFC 3 and FLAG ‐ FEN 1 in SW 480 and HT 29 cells simultaneously transduced with their cDNA s. Arrows and arrowheads indicate FLAG ‐tagged or endogenous proteins, respectively. E, MTT assay was performed for SW 480 and HT 29 cells transduced with mock vector or PCNA , RFC 3 and FEN 1 cDNA . Error bars, SD . P = .76 (*) and .090 ( † )
Techniques Used: Over Expression, Western Blot, MTT Assay, Transfection, Transduction, Plasmid Preparation
18) Product Images from "GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer"
Article Title: GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer
Journal: Cancer & Metabolism
doi: 10.1186/2049-3002-2-11

Figure Legend Snippet: GLUT3 is induced upon EMT. (A) H2122 cells were stimulated with 10 ng/ml TGF-β for the indicated time points, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 4) of mRNA expression ratios between TGF-β treated and non-treated conditions. (B) H727 or H2009 cell populations stably expressing a control plasmid (ctl), SNAIL, or ZEB1 were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (C) H727 or H2009 stable cell populations were lysed with RIPA buffer to prepare protein extracts and to analyze the expression of the indicated proteins by Western blot.
Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Expressing, Stable Transfection, Plasmid Preparation, CTL Assay, Western Blot

Figure Legend Snippet: GLUT3 is strongly expressed in mesenchymal liver tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). N.D. , not detected. (B) Hep3B cells were stimulated with 10 ng/ml TGF-β for 72 h, after which the cells were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA expression relative to the untreated conditions (set to 1).
Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Expressing

Figure Legend Snippet: GLUT3 is strongly expressed in mesenchymal lung tumor cells. (A) The indicated cell lines were lysed for RNA preparation followed by reverse transcription. The cDNA was amplified by real-time PCR using probes specific for the indicated genes or internal controls. Data show means ± s.d. ( n = 3) of mRNA or mature microRNA expression relative to the cell line expressing the least amount of the same gene (set to 1). (B) The indicated cells were stained to analyze the expression of GLUT3 or E-cadherin by immunocytochemistry. Scale bar, 100 μm. In (A) and (B) , the dashed line indicates the separation between the mesenchymal and the epithelial groups of cells.
Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Expressing, Staining, Immunocytochemistry
19) Product Images from "A MT1-MMP/NF-?B signaling axis as a checkpoint controller of COX-2 expression in CD133(+) U87 glioblastoma cells"
Article Title: A MT1-MMP/NF-?B signaling axis as a checkpoint controller of COX-2 expression in CD133(+) U87 glioblastoma cells
Journal: Journal of Neuroinflammation
doi: 10.1186/1742-2094-6-8

Figure Legend Snippet: Cell-based evidence that MT1-MMP directly regulates COX-2 expression in U87 glioma cell lines . Monolayers or neurospheres from glioblastoma-derived cells were either Mock-transfected, transfected with a cDNA plasmid encoding MT1-MMP (Wt), or transfected with an siRNA (Si) against MT1-MMP as described in the Methods section. (A) Gelatin zymography was performed to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western-blotting and COX-2 or GAPDH immunodetection. (B) Total RNA was isolated from monolayers (white bars) or neurospheres (black bars) of U87 Mock-transfected cells, or from U87 cells transfected with MT1-MMP cDNA or siRNA against MT1-MMP, and reverse-transcribed as described in the Methods section. Quantitative PCR was performed in order to monitor COX-2 gene expression levels.
Techniques Used: Expressing, Derivative Assay, Transfection, Plasmid Preparation, Zymography, Isolation, SDS Page, Western Blot, Immunodetection, Real-time Polymerase Chain Reaction

Figure Legend Snippet: MT1-MMP-mediated regulation of COX-2 gene and protein expression is independent from MT1-MMP's catalytic functions . (A) Cell lysates were isolated from Mock-transfected, or U87 glioma cells that had been transiently transfected with a cDNA plasmid encoding MT1-MMP, which were subsequently treated (or not) with 10 μM Ilomastat (Ilo). SDS-PAGE was performed (20 μg protein/well), followed by Western-blotting and COX-2 or MT1-MMP immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells. (B) Total RNA was extracted from the above described cell conditions and gene expression levels assessed by qRT-PCR for COX-2 in the absence (white bars) or the presence of Ilomastat (black bars).
Techniques Used: Expressing, Isolation, Transfection, Plasmid Preparation, SDS Page, Western Blot, Immunodetection, Zymography, Quantitative RT-PCR
20) Product Images from "Endoplasmic reticulum stress in adipose tissue augments lipolysis"
Article Title: Endoplasmic reticulum stress in adipose tissue augments lipolysis
Journal: Journal of Cellular and Molecular Medicine
doi: 10.1111/jcmm.12384

Figure Legend Snippet: Male Balb/c mice were injected with either control buffer (Con) or tunicamycin (Tun). After 24 hrs, the epididymal fat pads were dissected and ( A ) total RNA was extracted and transcribed to cDNA. Relative mRNA levels of the indicated genes were quantified using real-time PCR and normalized to 18S rRNA. The value obtained for the control for each gene was assigned a value of 1. The value obtained following injection with tunicamycin was expressed relative to the control value. Results represent the mean ± SD of n = 3–8 experiments. ▪ Control, □ Tunicamycin. ( B ) Equal amounts of protein were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing the indicated proteins. For IRE1α, GRP78 and GAPDH intervening lanes have been removed. ( C – E ) The intensities of the bands in ( B ) were quantified from n = 3–6 experiments. The control values were set to 1. The values obtained from tunicamycin treated mice were expressed relative to the control. * Indicates P
Techniques Used: Mouse Assay, Injection, Real-time Polymerase Chain Reaction, SDS Page

Figure Legend Snippet: Subcutaneous white adipose tissue was obtained during surgery from burned patients (burn) or non-burned patients undergoing elective surgery (non-burn). ( A ) Total RNA was extracted from adipose tissue obtained from non-burned ( n = 3–4) and burned patients ( n = 5–8) and transcribed to cDNA. Real-time quantitative PCR was performed to evaluate the relative mRNA levels of the indicated genes that were normalized to IDH1 mRNA. The value obtained from one non-burn sample was set to 1. The values obtained from all other samples were expressed relative to the non-burn sample. ▪ Non-burn, □ Burn. ( B , Top and Bottom Panels ) Adipose tissue was homogenized and equal amounts of total protein in the tissue homogenates were resolved by SDS-PAGE followed by immunoblotting using antibodies recognizing GRP78 and alpha/beta tubulin. ( C ) The intensities of the bands corresponding to GRP78 in ( B ) were quantified. ( D ) Human adipocytes (non-burn) were isolated and cultured either in the presence or absence of tunicamycin (5 μg/ml) at 37°C for the indicated times. The culture media was collected and the concentration of free glycerol determined. To account for the variability between patients in basal lipolysis rates, the amount of glycerol released from control cells from each patient and at each time-point was given a value of 100%. The amount of glycerol released from adipocytes in the presence of tunicamycin is expressed as a percentage of the value obtained from the control cells. Results represent the mean ± SD from n = 3 female patients ages 48 and 61 years, ▪ Control, □ Tunicamycin. * indicates P
Techniques Used: Real-time Polymerase Chain Reaction, SDS Page, Isolation, Cell Culture, Concentration Assay
Related Articles
Amplification:Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the Article Title: Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells Article Snippet: Total RNA of control cells and of nucleofected cells was extracted with TRIzol® Reagent (Invitrogen™ Life Technologies) and was reverse-transcribed with the Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Positive Control:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. Synthesized:Article Title: The Deacetylase Sirt6 Activates the Acetyltransferase GCN5 and Suppresses Hepatic Gluconeogenesis Article Snippet: Total RNA was isolated from cells or pulverized liver using Trizol (Invitrogen). .. For Q-RT-PCR analysis, cDNA was synthesized from 2 μg RNA using random primers and Pyrolysis Gas Chromatography:Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: The mRNA expression of PPARGC1 alpha (PGC-1α), citrate synthase (CS), 5-aminolevulinate synthase, (ALAS), cytochrome c , (cyt. c ), pyruvate dehydrogenase kinase 4 (PDK4), Drosha, DiGeorge syndrome critical region gene 8 (DGCR8) and Dicer were quantified using 7300 Real-time PCR System (Applied Biosystems Inc., Foster City, CA) and SYBR® Green chemistry (PerfeCT a SYBR® Green Supermix, ROX, Quanta BioSciences, Gaithersburg, MD) as previously described . .. First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Immunostaining:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: Total RNA was isolated from NSCs in the same culture conditions as those used in immunostaining. .. Real-time Polymerase Chain Reaction:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: For quantitative real-time PCR of IL-17R, specific primers were generated as follows: IL-17RrealF: 5′-AGGTCCAGCCCTTCTTCAGCA-3′, IL-17RrealR: 5′-GCTTGGGAACTGTGGTATTTGA- -GATTA-3′. .. Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits Interferon-Gamma Production in B Cells and Suppresses Colitis in Mice Article Snippet: Analysis of Abs in sera collected from 12- to 18-month-old mice was performed by ELISA using IL-4, IL-10, IL-17, and IFN-γ ELISA Kits (Alpha Diagnostic International) according to the manufacturer’s instructions. .. RNA was isolated using an RNeasy Plus Micro Kit (Qiagen) and reverse-transcribed using a Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury Article Snippet: Paragraph title: Determination of mRNA Expression for HO-1, HO-2, HIF-1α, NF-κB, COX-2, iNOS, TNF-α, IL-1β, SOD-2, and GPx-1 in Rat Gastric Mucosa by Real-Time Polymerase Chain Reaction (qPCR) ... Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: RNA expression was quantified using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, California). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: Paragraph title: Real-time PCR for mRNA expression analyses ... The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: The mRNA expression of PPARGC1 alpha (PGC-1α), citrate synthase (CS), 5-aminolevulinate synthase, (ALAS), cytochrome c , (cyt. c ), pyruvate dehydrogenase kinase 4 (PDK4), Drosha, DiGeorge syndrome critical region gene 8 (DGCR8) and Dicer were quantified using 7300 Real-time PCR System (Applied Biosystems Inc., Foster City, CA) and SYBR® Green chemistry (PerfeCT a SYBR® Green Supermix, ROX, Quanta BioSciences, Gaithersburg, MD) as previously described . .. First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms Article Snippet: Paragraph title: Quantitative PCR ... RNA was reverse-transcribed using Microarray:Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms Article Snippet: RNA was reverse-transcribed using Quantitation Assay:Article Title: Article Snippet: AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a Expressing:Article Title: Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation Article Snippet: Paragraph title: Gene Expression Profiling by PCR Array ... Total RNA (1 μg) in a 20-μl final volume was reverse-transcribed using the Article Title: Expression of SV2 isoforms during rodent brain development Article Snippet: One μg of total RNA was used to synthesize cDNA with the Applied Biosystems Article Title: The Deacetylase Sirt6 Activates the Acetyltransferase GCN5 and Suppresses Hepatic Gluconeogenesis Article Snippet: Paragraph title: Gene Expression Analysis ... For Q-RT-PCR analysis, cDNA was synthesized from 2 μg RNA using random primers and Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits Interferon-Gamma Production in B Cells and Suppresses Colitis in Mice Article Snippet: Analysis of Abs in sera collected from 12- to 18-month-old mice was performed by ELISA using IL-4, IL-10, IL-17, and IFN-γ ELISA Kits (Alpha Diagnostic International) according to the manufacturer’s instructions. .. RNA was isolated using an RNeasy Plus Micro Kit (Qiagen) and reverse-transcribed using a Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury Article Snippet: Paragraph title: Determination of mRNA Expression for HO-1, HO-2, HIF-1α, NF-κB, COX-2, iNOS, TNF-α, IL-1β, SOD-2, and GPx-1 in Rat Gastric Mucosa by Real-Time Polymerase Chain Reaction (qPCR) ... Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using Article Title: Article Snippet: AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a Article Title: Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch Article Snippet: The RNA and nontemplate (i.e., H2 O instead of RNA) samples were reverse transcribed using a Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: Paragraph title: 2.7.Analysis of mRNA expression ... One microgram of total RNA was converted to cDNA using the Applied Biosystems Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: Paragraph title: Real-time PCR for mRNA expression analyses ... The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: Paragraph title: mRNA Expression analyses ... First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES Article Snippet: CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the Cell Fractionation:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Paragraph title: Cell fractionation, Northern and western blotting ... 1 μg total RNA was reverse transcribed using the Western Blot:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Paragraph title: Cell fractionation, Northern and western blotting ... 1 μg total RNA was reverse transcribed using the Activated Clotting Time Assay:Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems Countercurrent Chromatography:Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems Transfection:Article Title: Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells Article Snippet: Paragraph title: Detection of human factor IX mRNA in transfected cells ... Total RNA of control cells and of nucleofected cells was extracted with TRIzol® Reagent (Invitrogen™ Life Technologies) and was reverse-transcribed with the Dissection:Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts Article Snippet: RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using Northern Blot:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Paragraph title: Cell fractionation, Northern and western blotting ... 1 μg total RNA was reverse transcribed using the Generated:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: For quantitative real-time PCR of IL-17R, specific primers were generated as follows: IL-17RrealF: 5′-AGGTCCAGCCCTTCTTCAGCA-3′, IL-17RrealR: 5′-GCTTGGGAACTGTGGTATTTGA- -GATTA-3′. .. Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Inhibition:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the Polymerase Chain Reaction:Article Title: Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation Article Snippet: Paragraph title: Gene Expression Profiling by PCR Array ... Total RNA (1 μg) in a 20-μl final volume was reverse-transcribed using the Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: For quantitative real-time PCR of IL-17R, specific primers were generated as follows: IL-17RrealF: 5′-AGGTCCAGCCCTTCTTCAGCA-3′, IL-17RrealR: 5′-GCTTGGGAACTGTGGTATTTGA- -GATTA-3′. .. Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury Article Snippet: Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using Article Title: Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch Article Snippet: The RNA and nontemplate (i.e., H2 O instead of RNA) samples were reverse transcribed using a Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma Article Snippet: Paragraph title: In-house PCR array and Primer Design ... Approximately 2 µg of total RNA was reverse transcribed to cDNA by the Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the Article Title: Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells Article Snippet: Total RNA of control cells and of nucleofected cells was extracted with TRIzol® Reagent (Invitrogen™ Life Technologies) and was reverse-transcribed with the Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES Article Snippet: Total RNA was collected from the cell pellet and extracted with TRI reagent (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. .. CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the Quantitative RT-PCR:Article Title: Article Snippet: Paragraph title: Quantitative RT-PCR to Analyze COX-2 mRNA ... AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a Article Title: p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells Article Snippet: Paragraph title: RNA isolation and qRT-PCR for mRNA detection ... 1 μg of DNAse-treated RNA (RQ1 RNase-Free DNase kit, Promega, Madison, WI, USA) was retrotranscribed with Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: At the end of the developmental toxicity screen, animals exposed to 0, 0.01, 0.1, 1 and 10 µM of E2 and EE2 using either the Liquid Handler or BioPrinter were collected and pooled to create 3 biological replicates for quantitative RT-PCR analysis. .. One microgram of total RNA was converted to cDNA using the Applied Biosystems Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy Article Snippet: Paragraph title: Real-time quantitative RT-PCR ... Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES Article Snippet: Total RNA was collected from the cell pellet and extracted with TRI reagent (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. .. CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the TaqMan Assay:Article Title: p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells Article Snippet: 1 μg of DNAse-treated RNA (RQ1 RNase-Free DNase kit, Promega, Madison, WI, USA) was retrotranscribed with In Vivo:Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy Article Snippet: Total RNA was isolated from in vivo samples using ISOGEN II (Nippongene) according to the manufacturer's instructions. .. Fluorescence:Article Title: CALCIUM OXALATE STONE FRAGMENT AND CRYSTAL PHAGOCYTOSIS BY HUMAN MACROPHAGES Article Snippet: CCL2, CCL3, CCL4, CCL22, IL-1Rα, IL-6, IL-8, S100a9, and GAPDH levels were analyzed by qRT-PCR. mRNA qRT-PCR was performed using the Isolation:Article Title: Role of Chronic Inflammation in Myopia Progression: Clinical Evidence and Experimental Validation Article Snippet: 2.6 Total RNA of sclera tissues was isolated using an RNeasy Mini kit (Qiagen) and used for PCR array analysis. .. Total RNA (1 μg) in a 20-μl final volume was reverse-transcribed using the Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. Article Title: The Deacetylase Sirt6 Activates the Acetyltransferase GCN5 and Suppresses Hepatic Gluconeogenesis Article Snippet: Total RNA was isolated from cells or pulverized liver using Trizol (Invitrogen). .. For Q-RT-PCR analysis, cDNA was synthesized from 2 μg RNA using random primers and Article Title: Glucocorticoid-Induced Leucine Zipper Inhibits Interferon-Gamma Production in B Cells and Suppresses Colitis in Mice Article Snippet: Analysis of Abs in sera collected from 12- to 18-month-old mice was performed by ELISA using IL-4, IL-10, IL-17, and IFN-γ ELISA Kits (Alpha Diagnostic International) according to the manufacturer’s instructions. .. RNA was isolated using an RNeasy Plus Micro Kit (Qiagen) and reverse-transcribed using a Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury Article Snippet: Expression of mRNA in gastric mucosa was determined by real-time PCR as described previously [ ]. .. Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using Article Title: Article Snippet: Phosphorylation of VEGFR-2 and VEGFR-3 on adult LECs (AdLECs, Lonza) treated with VEGF-C or VEGF-D variants at 200 ng/ml was analyzed as described previously ( ). .. AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a Article Title: p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells Article Snippet: Paragraph title: RNA isolation and qRT-PCR for mRNA detection ... 1 μg of DNAse-treated RNA (RQ1 RNase-Free DNase kit, Promega, Madison, WI, USA) was retrotranscribed with Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: Briefly, total RNA was isolated from groups of 120 hpf larval zebrafish using RNAzol® RT (Molecular Research Center, Inc., Cincinnati, OH). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy Article Snippet: Total RNA was isolated from in vivo samples using ISOGEN II (Nippongene) according to the manufacturer's instructions. .. Negative Control:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. Article Title: Cell-autonomous regulation of fast troponin T pre-mRNA alternative splicing in response to mechanical stretch Article Snippet: The RNA and nontemplate (i.e., H2 O instead of RNA) samples were reverse transcribed using a Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a Mouse Assay:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: RNAs isolated from primary oligodendrocytes ( > 93% GalC+ ) of wild type B6 mice served as IL-17R positive control [ ], and of IL-17R-deficient mice (the Jackson Laboratory) as negative control. .. Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms Article Snippet: RNA was reverse-transcribed using Reverse Transcription Polymerase Chain Reaction:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the Blocking Assay:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: For detection of DIG-labeled probes, the membrane was briefly washed in washing buffer (0.1M Maleic Acid, 0.15M NaCl, pH 7,5 and 0.3% Tween, pH7.5) and blocked with 0.1M Maleic Acid, 0.15M NaCl, 1% Blocking Reagent (Roche) at room temperature. .. 1 μg total RNA was reverse transcribed using the Chloramphenicol Acetyltransferase Assay:Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems Purification:Article Title: Expression of SV2 isoforms during rodent brain development Article Snippet: Paragraph title: RNA purification and quantification ... One μg of total RNA was used to synthesize cDNA with the Applied Biosystems Article Title: Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury Article Snippet: Expression of mRNA in gastric mucosa was determined by real-time PCR as described previously [ ]. .. Briefly, RNA was isolated from gastric mucosal biopsies stored at −80 °C using GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland) and reversed transcription to cDNA was performed using Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: After 24 h of pre-incubation, cells were treated with obtained leaves and tuber extracts and incubated for 24 h. Then, samples were collected, and total RNA was extracted from HaCaT and fibroblast cells using a EURx Universal RNA Purification Kit according to the manufacturer’s protocol. .. The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the SDS Page:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: For western blot, 1/50 of total lysates volume was loaded onto a 10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore) followed by incubation with primary and HRP-conjugated secondary antibodies and ECL detection. .. 1 μg total RNA was reverse transcribed using the Software:Article Title: Expression of SV2 isoforms during rodent brain development Article Snippet: One μg of total RNA was used to synthesize cDNA with the Applied Biosystems Article Title: Article Snippet: AdLECs were serum-starved overnight and then exposed to VEGF-C or VEGF-D variants (at 100 ng/ml, unless stated otherwise) before isolation of total RNA using an RNeasy minikit (Qiagen, Valencia, CA) and preparation of cDNA with a Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the SYBR Green Assay:Article Title: Inhibitory effect of IL-17 on neural stem cell proliferation and neural cell differentiation Article Snippet: For quantitative real-time PCR of IL-17R, specific primers were generated as follows: IL-17RrealF: 5′-AGGTCCAGCCCTTCTTCAGCA-3′, IL-17RrealR: 5′-GCTTGGGAACTGTGGTATTTGA- -GATTA-3′. .. Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: Inhibition of de novo RNA transcription by ActD was validated by quantitative PCR. .. 1 μg total RNA was reverse transcribed using the Article Title: miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice Article Snippet: The mRNA expression of PPARGC1 alpha (PGC-1α), citrate synthase (CS), 5-aminolevulinate synthase, (ALAS), cytochrome c , (cyt. c ), pyruvate dehydrogenase kinase 4 (PDK4), Drosha, DiGeorge syndrome critical region gene 8 (DGCR8) and Dicer were quantified using 7300 Real-time PCR System (Applied Biosystems Inc., Foster City, CA) and SYBR® Green chemistry (PerfeCT a SYBR® Green Supermix, ROX, Quanta BioSciences, Gaithersburg, MD) as previously described . .. First-strand cDNA synthesis from 1 µg of total RNA was performed with random primers using a RNA Extraction:Article Title: Expression of SV2 isoforms during rodent brain development Article Snippet: RNA extraction was performed with the RNeasy (Qiagen®, Venlo, Netherlands). .. One μg of total RNA was used to synthesize cDNA with the Applied Biosystems RNA Expression:Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). .. One microgram of total RNA was converted to cDNA using the Applied Biosystems In Vitro:Article Title: Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy Article Snippet: In vitro RNA samples were prepared as mentioned above. .. Homogenization:Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using Incubation:Article Title: Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response Article Snippet: For western blot, 1/50 of total lysates volume was loaded onto a 10% SDS-PAGE and transferred to an Immobilon-P PVDF membrane (Millipore) followed by incubation with primary and HRP-conjugated secondary antibodies and ECL detection. .. 1 μg total RNA was reverse transcribed using the Article Title: Antioxidant activity and cytotoxicity of Jerusalem artichoke tubers and leaves extract on HaCaT and BJ fibroblast cells Article Snippet: After 24 h of pre-incubation, cells were treated with obtained leaves and tuber extracts and incubated for 24 h. Then, samples were collected, and total RNA was extracted from HaCaT and fibroblast cells using a EURx Universal RNA Purification Kit according to the manufacturer’s protocol. .. The reverse transcription (RT) reaction was performed at a final volume 20 μL with 1 μg of RNA (as a cDNA template) using the Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using Concentration Assay:Article Title: Improved detection of gene-microbe interactions in the mouse skin microbiota using high-resolution QTL mapping of 16S rRNA transcripts Article Snippet: An additional 2-h room temperature incubation step was included after homogenization in order to increase the nucleic acids’ dissolution in the RLT buffer. .. RNA was treated with DNase (RNase-Free DNase Qiagen, stock solution concentration) for 15 min, twice. cDNA synthesis was performed using Article Title: Systematic evaluation of medium-throughput mRNA abundance platforms Article Snippet: RNA was reverse-transcribed using CTG Assay:Article Title: Optimizing multi-dimensional high throughput screening using zebrafish Article Snippet: One microgram of total RNA was converted to cDNA using the Applied Biosystems Variant Assay:Article Title: A Novel Predictive Equation for Potential Diagnosis of Cholangiocarcinoma Article Snippet: Approximately 2 µg of total RNA was reverse transcribed to cDNA by the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. .. Approximately 2 µg of total RNA was reverse transcribed to cDNA by the |