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  • 99
    Qiagen rneasy
    Amino acid sequence alignment around His-171, GEF activity, and gene expression of FGD3. (A) His-171 of bovine FGD3 and the flanking region is well conserved among mammalian species. The RhoGEF domain corresponds to the region from His-164 to Ala-344 of bovine FGD3. (B) NIH3T3 cells were transfected with the bicistronic expression plasmid encoding HA-tagged wild or mutant bovine FGD3(SA) and V5-tagged Cdc42. FGD3(SA) denotes FGD3 with two serines in the destruction motif (Ser-83 and Ser-87 of bovine FGD3) replaced by alanines [ 24 ]. After 48 hours, cell extracts were prepared and submitted to the GST–CRIB pull-down assay. A portion of cell extracts and the pull-down products were subjected to SDS-PAGE followed by immunoblotting to detect Cdc42-V5 and FGD3(SA)-HA. The experiment was repeated two more times, and the results are shown in S4 Fig . (C) <t>RNA</t> was extracted using RNAiso Plus (Takara) from tissues of a 1-month-old Holstein calf. Primary chondrocytes were prepared from the ear cartilage and cultured as micromass. Five days later, RNA was extracted from the micromass using an <t>RNeasy</t> kit (QIAGEN, Valencia, CA, USA). RT-PCR was performed using a standard method and PCR primers given in S8 Table . PCR products were resolved in a 2% agarose gel. Lane M, 100-bp DNA ladder; lane 1, bone marrow just under the growth plate of a femur; lane 2, growth plate cartilage of the femur; lane 3, ear cartilage; lane 4, micromass culture from the ear cartilage. (D) In situ hybridization of Fgd3 on the femur of a 3-week-old C57BL/6 mouse. Sense and antisense RNA probes correspond to nt. 129–956 of the mouse Fgd3 cDNA (NM_015759).
    Rneasy, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 28471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy/product/Qiagen
    Average 99 stars, based on 28471 article reviews
    Price from $9.99 to $1999.99
    rneasy - by Bioz Stars, 2020-07
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    98
    Qiagen rneasy maxi
    Indel Formation in CD34+ HSPCs Nucleofected with Modified Cas9 <t>mRNAs</t> CD34+ HSPCs were nucleofected with 3 μg of the indicated Cas9 mRNA and 2 μg IL2RGlocus MS-sgRNA. 6 μg Cas9 RNP complexed to 3.2 μg IL2RGlocus MS-sgRNA was nucleofected for comparison. ARCA 5meC/Ψ is our previously published Cas9 mRNA 6 and was also included for comparison. Cells were isolated after 4 days, and indel formation was assessed by TIDE analysis. Bars represent mean ± SEM of at least 5 independent transfections. White and gray bars indicate <t>RNeasy</t> and HPLC-purified mRNAs, respectively. sgRNA complexed to Cas9 RNP was included as a control. ***p
    Rneasy Maxi, supplied by Qiagen, used in various techniques. Bioz Stars score: 98/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy maxi/product/Qiagen
    Average 98 stars, based on 98 article reviews
    Price from $9.99 to $1999.99
    rneasy maxi - by Bioz Stars, 2020-07
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    99
    Qiagen rneasy midi
    Indel Formation in CD34+ HSPCs Nucleofected with Modified Cas9 <t>mRNAs</t> CD34+ HSPCs were nucleofected with 3 μg of the indicated Cas9 mRNA and 2 μg IL2RGlocus MS-sgRNA. 6 μg Cas9 RNP complexed to 3.2 μg IL2RGlocus MS-sgRNA was nucleofected for comparison. ARCA 5meC/Ψ is our previously published Cas9 mRNA 6 and was also included for comparison. Cells were isolated after 4 days, and indel formation was assessed by TIDE analysis. Bars represent mean ± SEM of at least 5 independent transfections. White and gray bars indicate <t>RNeasy</t> and HPLC-purified mRNAs, respectively. sgRNA complexed to Cas9 RNP was included as a control. ***p
    Rneasy Midi, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy midi/product/Qiagen
    Average 99 stars, based on 137 article reviews
    Price from $9.99 to $1999.99
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    99
    Qiagen rneasy mini
    <t>RNA</t> extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the <t>RNeasy®</t> Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Rneasy Mini, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy mini/product/Qiagen
    Average 99 stars, based on 4603 article reviews
    Price from $9.99 to $1999.99
    rneasy mini - by Bioz Stars, 2020-07
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    99
    Qiagen rneasy ffpe
    <t>RNA</t> extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the <t>RNeasy®</t> Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Rneasy Ffpe, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy ffpe/product/Qiagen
    Average 99 stars, based on 129 article reviews
    Price from $9.99 to $1999.99
    rneasy ffpe - by Bioz Stars, 2020-07
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    99
    Qiagen rneasy mini kit rneasy mini
    <t>RNA</t> extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the <t>RNeasy®</t> Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Rneasy Mini Kit Rneasy Mini, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy mini kit rneasy mini/product/Qiagen
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rneasy mini kit rneasy mini - by Bioz Stars, 2020-07
    99/100 stars
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    99
    Qiagen rneasy 96
    <t>RNA</t> extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the <t>RNeasy®</t> Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Rneasy 96, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 82 article reviews
    Price from $9.99 to $1999.99
    rneasy 96 - by Bioz Stars, 2020-07
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    92
    Promega rneasy
    <t>RNA</t> extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the <t>RNeasy®</t> Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Rneasy, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy/product/Promega
    Average 92 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    rneasy - by Bioz Stars, 2020-07
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    Image Search Results


    Amino acid sequence alignment around His-171, GEF activity, and gene expression of FGD3. (A) His-171 of bovine FGD3 and the flanking region is well conserved among mammalian species. The RhoGEF domain corresponds to the region from His-164 to Ala-344 of bovine FGD3. (B) NIH3T3 cells were transfected with the bicistronic expression plasmid encoding HA-tagged wild or mutant bovine FGD3(SA) and V5-tagged Cdc42. FGD3(SA) denotes FGD3 with two serines in the destruction motif (Ser-83 and Ser-87 of bovine FGD3) replaced by alanines [ 24 ]. After 48 hours, cell extracts were prepared and submitted to the GST–CRIB pull-down assay. A portion of cell extracts and the pull-down products were subjected to SDS-PAGE followed by immunoblotting to detect Cdc42-V5 and FGD3(SA)-HA. The experiment was repeated two more times, and the results are shown in S4 Fig . (C) RNA was extracted using RNAiso Plus (Takara) from tissues of a 1-month-old Holstein calf. Primary chondrocytes were prepared from the ear cartilage and cultured as micromass. Five days later, RNA was extracted from the micromass using an RNeasy kit (QIAGEN, Valencia, CA, USA). RT-PCR was performed using a standard method and PCR primers given in S8 Table . PCR products were resolved in a 2% agarose gel. Lane M, 100-bp DNA ladder; lane 1, bone marrow just under the growth plate of a femur; lane 2, growth plate cartilage of the femur; lane 3, ear cartilage; lane 4, micromass culture from the ear cartilage. (D) In situ hybridization of Fgd3 on the femur of a 3-week-old C57BL/6 mouse. Sense and antisense RNA probes correspond to nt. 129–956 of the mouse Fgd3 cDNA (NM_015759).

    Journal: PLoS Genetics

    Article Title: Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

    doi: 10.1371/journal.pgen.1005433

    Figure Lengend Snippet: Amino acid sequence alignment around His-171, GEF activity, and gene expression of FGD3. (A) His-171 of bovine FGD3 and the flanking region is well conserved among mammalian species. The RhoGEF domain corresponds to the region from His-164 to Ala-344 of bovine FGD3. (B) NIH3T3 cells were transfected with the bicistronic expression plasmid encoding HA-tagged wild or mutant bovine FGD3(SA) and V5-tagged Cdc42. FGD3(SA) denotes FGD3 with two serines in the destruction motif (Ser-83 and Ser-87 of bovine FGD3) replaced by alanines [ 24 ]. After 48 hours, cell extracts were prepared and submitted to the GST–CRIB pull-down assay. A portion of cell extracts and the pull-down products were subjected to SDS-PAGE followed by immunoblotting to detect Cdc42-V5 and FGD3(SA)-HA. The experiment was repeated two more times, and the results are shown in S4 Fig . (C) RNA was extracted using RNAiso Plus (Takara) from tissues of a 1-month-old Holstein calf. Primary chondrocytes were prepared from the ear cartilage and cultured as micromass. Five days later, RNA was extracted from the micromass using an RNeasy kit (QIAGEN, Valencia, CA, USA). RT-PCR was performed using a standard method and PCR primers given in S8 Table . PCR products were resolved in a 2% agarose gel. Lane M, 100-bp DNA ladder; lane 1, bone marrow just under the growth plate of a femur; lane 2, growth plate cartilage of the femur; lane 3, ear cartilage; lane 4, micromass culture from the ear cartilage. (D) In situ hybridization of Fgd3 on the femur of a 3-week-old C57BL/6 mouse. Sense and antisense RNA probes correspond to nt. 129–956 of the mouse Fgd3 cDNA (NM_015759).

    Article Snippet: RNA was extracted from testes of three heterozygous calves using RNeasy (QIAGEN).

    Techniques: Sequencing, Activity Assay, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Pull Down Assay, SDS Page, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Situ Hybridization

    Comparison of plate formats and RNA extraction kits. (A) Serial dilutions of SARS-CoV-2 synthetic RNA standards from Twist Biosci (in copies/μl of the standard added to the RT-qPCR reaction) run in parallel on separate BioRad CFX 96-well (20 μl reactions) or 384-well (10 μl reactions) real-time PCR systems using the Norgen COVID-19 RT-qPCR detection module. Mean +/- range of two independent tests. (B-C) Analysis of four negative and four positive patient samples extracted with either the Qiagen RNeasy or Norgen RNA isolation kits (B) or two positive patient samples extracted with the Norgen, Invitrogen Purelink or BGI RNA isolation kits (C) using the Norgen RT-qPCR detection system and N1, N2 or human control (Rnase P) primers. Samples L015, L018 and L019 are the mean +/- range of technical duplicates run independently on two separate plates, other samples were analyzed once, although L028 and L029 are rerun in Fig. 2a . In (B) a paired t-test was used to compare Norgen vs . Qiagen extractions.

    Journal: bioRxiv

    Article Title: Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

    doi: 10.1101/2020.05.12.092387

    Figure Lengend Snippet: Comparison of plate formats and RNA extraction kits. (A) Serial dilutions of SARS-CoV-2 synthetic RNA standards from Twist Biosci (in copies/μl of the standard added to the RT-qPCR reaction) run in parallel on separate BioRad CFX 96-well (20 μl reactions) or 384-well (10 μl reactions) real-time PCR systems using the Norgen COVID-19 RT-qPCR detection module. Mean +/- range of two independent tests. (B-C) Analysis of four negative and four positive patient samples extracted with either the Qiagen RNeasy or Norgen RNA isolation kits (B) or two positive patient samples extracted with the Norgen, Invitrogen Purelink or BGI RNA isolation kits (C) using the Norgen RT-qPCR detection system and N1, N2 or human control (Rnase P) primers. Samples L015, L018 and L019 are the mean +/- range of technical duplicates run independently on two separate plates, other samples were analyzed once, although L028 and L029 are rerun in Fig. 2a . In (B) a paired t-test was used to compare Norgen vs . Qiagen extractions.

    Article Snippet: We compare the RNA isolation systems from both companies alongside the Qiagen RNeasy and Invitrogen Purelink systems, both of which are routinely used in research labs, and the former of which has been shown to provide only modestly reduced recovery compared to the CDC approved QIAamp Viral RNA kit ( ).

    Techniques: RNA Extraction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation

    NFI protein and mRNA levels do not change following TGF-β treatment of HKc/HPV16. (A) Total NFI protein levels were determined by Western analysis. Nuclear extract (40 μg of protein) from TGF-β-sensitive HKc/HPV16 treated with and without 40 pM TGF-β for 48 h was separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and probed with an anti-NFI antibody (Santa Cruz). Molecular mass markers are shown on the right; arrows pointing to NFI bands are on the left. (B) mRNA expression of each of the four NFI family members (NFIA, NFIB, NFIC, and NFIX) was determined by real-time PCR. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 treated with (gray) and without (black) 40 pM TGF-β for 46 h. Expression was determined using primers specific for each NFI family member, compared with a control set of primers, and expressed as fold induction. The average of two experiments for each family member is shown.

    Journal: Journal of Virology

    Article Title: NFI-Ski Interactions Mediate Transforming Growth Factor ? Modulation of Human Papillomavirus Type 16 Early Gene Expression

    doi: 10.1128/JVI.78.8.3953-3964.2004

    Figure Lengend Snippet: NFI protein and mRNA levels do not change following TGF-β treatment of HKc/HPV16. (A) Total NFI protein levels were determined by Western analysis. Nuclear extract (40 μg of protein) from TGF-β-sensitive HKc/HPV16 treated with and without 40 pM TGF-β for 48 h was separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and probed with an anti-NFI antibody (Santa Cruz). Molecular mass markers are shown on the right; arrows pointing to NFI bands are on the left. (B) mRNA expression of each of the four NFI family members (NFIA, NFIB, NFIC, and NFIX) was determined by real-time PCR. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 treated with (gray) and without (black) 40 pM TGF-β for 46 h. Expression was determined using primers specific for each NFI family member, compared with a control set of primers, and expressed as fold induction. The average of two experiments for each family member is shown.

    Article Snippet: RNA was collected using RNeasy columns (Qiagen).

    Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

    TGF-β inhibition of the HPV16 URR is overcome by overexpression of either NFI or Ski. (A) Effects of NFI family member overexpression on TGF-β modulation of the HPV16 URR. pGL3/URR and pCMV vectors expressing each of the NFI family members were cotransfected into TGF-β-sensitive HKc/HPV16 using Transfast (Promega). Luciferase activity was determined after treatment with and without 40 pM TGF-β for 42 h, 68 h posttransfection. TGF-β inhibition resulting from transfection of an empty vector (black bar) is compared with the percent TGF-β inhibition upon overexpression of each NFI family member (gray bars). (B) Effects of Ski overexpression on TGF-β modulation of the HPV16 URR. pGL3/URR and a pcDNA3.1 vector expressing Ski were cotransfected and analyzed as described above (A). (C) NFI and Ski protein levels were determined by Western analysis. Lysates from TGF-β-sensitive HKc/HPV16 cotransfected with pGL3/URR and pCMV vectors expressing each NFI family member, empty pCMV vector, Ski, or an empty pcDNA3.1 vector were separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and probed with an anti-NFI or an anti-Ski antibody. Molecular mass markers are shown on the right with arrows pointing to three NFI bands (top panel) or Ski (bottom panel). (D) mRNA expression of each NFI family member and Ski was determined by real-time PCR analysis. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 cotransfected with pGL3/URR and pCMV vectors expressing each NFI family member or a pcDNA vector expressing Ski. Expression was determined using primers specific for each NFI family member or Ski and is given as fold induction over empty vector.

    Journal: Journal of Virology

    Article Title: NFI-Ski Interactions Mediate Transforming Growth Factor ? Modulation of Human Papillomavirus Type 16 Early Gene Expression

    doi: 10.1128/JVI.78.8.3953-3964.2004

    Figure Lengend Snippet: TGF-β inhibition of the HPV16 URR is overcome by overexpression of either NFI or Ski. (A) Effects of NFI family member overexpression on TGF-β modulation of the HPV16 URR. pGL3/URR and pCMV vectors expressing each of the NFI family members were cotransfected into TGF-β-sensitive HKc/HPV16 using Transfast (Promega). Luciferase activity was determined after treatment with and without 40 pM TGF-β for 42 h, 68 h posttransfection. TGF-β inhibition resulting from transfection of an empty vector (black bar) is compared with the percent TGF-β inhibition upon overexpression of each NFI family member (gray bars). (B) Effects of Ski overexpression on TGF-β modulation of the HPV16 URR. pGL3/URR and a pcDNA3.1 vector expressing Ski were cotransfected and analyzed as described above (A). (C) NFI and Ski protein levels were determined by Western analysis. Lysates from TGF-β-sensitive HKc/HPV16 cotransfected with pGL3/URR and pCMV vectors expressing each NFI family member, empty pCMV vector, Ski, or an empty pcDNA3.1 vector were separated on an SDS-12% polyacrylamide gel, transferred to nitrocellulose, and probed with an anti-NFI or an anti-Ski antibody. Molecular mass markers are shown on the right with arrows pointing to three NFI bands (top panel) or Ski (bottom panel). (D) mRNA expression of each NFI family member and Ski was determined by real-time PCR analysis. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 cotransfected with pGL3/URR and pCMV vectors expressing each NFI family member or a pcDNA vector expressing Ski. Expression was determined using primers specific for each NFI family member or Ski and is given as fold induction over empty vector.

    Article Snippet: RNA was collected using RNeasy columns (Qiagen).

    Techniques: Inhibition, Over Expression, Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction

    TGF-β treatment of HKc/HPV16 and HKc/DR decreases nuclear Ski levels, and Ski interacts with NFI. (A) Ski levels were demonstrated by Western analysis (lanes 1 to 4). Nuclear extract (30 μg of protein) from TGF-β-sensitive HKc/HPV16 and TGF-β-resistant HKc/DR treated with and without 40 pM TGF-β for 48 h was separated on an SDS-10% polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with an anti-Ski antibody (Cascade Bioscience), which detects Ski products (brackets) ranging from 95 to 115 kDa. Endogenous Ski coimmunoprecipitated with NFI (lanes 5 to 8). Nuclear extract (850 μg of protein) from TGF-β-sensitive HKc/HPV16 and TGF-β-resistant HKc/DR treated with and without 40 pM TGF-β for 24 h was incubated (2 h, 25°C) with 5 μg of anti-NFI antibody preincubated with protein G agarose. After washing, the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and probed for Ski as described above. Molecular mass markers are noted on the right. (B) mRNA expression of Ski was determined by real-time PCR. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 treated with (gray) and without (black) 40 pM TGF-β for 46 h. Expression was determined using primers specific for Ski, compared with a control set of primers, and expressed as fold induction. The average of two experiments for each family member is shown.

    Journal: Journal of Virology

    Article Title: NFI-Ski Interactions Mediate Transforming Growth Factor ? Modulation of Human Papillomavirus Type 16 Early Gene Expression

    doi: 10.1128/JVI.78.8.3953-3964.2004

    Figure Lengend Snippet: TGF-β treatment of HKc/HPV16 and HKc/DR decreases nuclear Ski levels, and Ski interacts with NFI. (A) Ski levels were demonstrated by Western analysis (lanes 1 to 4). Nuclear extract (30 μg of protein) from TGF-β-sensitive HKc/HPV16 and TGF-β-resistant HKc/DR treated with and without 40 pM TGF-β for 48 h was separated on an SDS-10% polyacrylamide gel. Proteins were transferred to nitrocellulose and probed with an anti-Ski antibody (Cascade Bioscience), which detects Ski products (brackets) ranging from 95 to 115 kDa. Endogenous Ski coimmunoprecipitated with NFI (lanes 5 to 8). Nuclear extract (850 μg of protein) from TGF-β-sensitive HKc/HPV16 and TGF-β-resistant HKc/DR treated with and without 40 pM TGF-β for 24 h was incubated (2 h, 25°C) with 5 μg of anti-NFI antibody preincubated with protein G agarose. After washing, the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and probed for Ski as described above. Molecular mass markers are noted on the right. (B) mRNA expression of Ski was determined by real-time PCR. RNA was collected using RNeasy columns (Qiagen) from TGF-β-sensitive HKc/HPV16 treated with (gray) and without (black) 40 pM TGF-β for 46 h. Expression was determined using primers specific for Ski, compared with a control set of primers, and expressed as fold induction. The average of two experiments for each family member is shown.

    Article Snippet: RNA was collected using RNeasy columns (Qiagen).

    Techniques: Western Blot, Incubation, Polyacrylamide Gel Electrophoresis, Expressing, Real-time Polymerase Chain Reaction

    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Journal: PLoS Pathogens

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    doi: 10.1371/journal.ppat.1002642

    Figure Lengend Snippet: 2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Article Snippet: For each time point, total cellular RNA was extracted using RNeasy kit (Qiagen).

    Techniques: Methylation, Incubation, In Vitro, Generated

    MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P

    Journal: Cell Death & Disease

    Article Title: Long non-coding RNA MUC5B-AS1 promotes metastasis through mutually regulating MUC5B expression in lung adenocarcinoma

    doi: 10.1038/s41419-018-0472-6

    Figure Lengend Snippet: MUC5B-AS1 increases the stability of MUC5B mRNA by forming a protective RNA duplex. a Schematic representation of the PCR amplification regions for overlapping (OL) and non-overlapping (non-OL) regions of MUC5B. We designed two pairs of primers to amplify the OL regions (OL1 and OL2) and non-OL (non-OL1 and non-OL2) regions of MUC5B, respectively. F forward primer, R reverse primer. b RT-PCR products of OL and non-OL regions of MUC5B. Total RNA samples were treated with RNAse A + T cocktail and then cleaned up RNA using RNeasy kits. RT-PCR was conducted using the primers to detect the OL and non-OL regions of the MUC5B mRNA. OL and non-OL regions of KRT7-AS were used as a positive control. c Stability of MUC5B mRNA over 12 h was measured by qRT-PCR relative to time 0 h after blocking new RNA synthesis with Actinomycin D (1 μg/mL; indicated with black arrow). H1299 cells with MUC5B-AS1 or empty vector stable expression were treated with 1 μg/mL ActD, and then harvested cells for RNA purification at 12 h after addition of ActD. Then, MUC5B mRNA stability were subsequently measured by qRT-PCR and were normalized against a synthesized exogenous reference λ polyA + RNA. Student’s t -test, * P

    Article Snippet: We then cleaned up RNA using RNeasy kits (QIAGEN, Madison, WI, USA) and carried out reverse transcription-polymerase chain reaction (RT-PCR) using the primers to detect the OL and non-OL regions of the MUC5B mRNA.

    Techniques: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Blocking Assay, Plasmid Preparation, Expressing, Purification, Synthesized

    RNA integrity values for material isolated from LCM samples, whole tissue slices and cell lines. RNA quality (RIN values) for LCM derived samples, whole tissue slices and cell lines isolated with the RNeasy or miRNeasy kits. Median RIN values are shown, whiskers indicate interquartile range

    Journal: BMC Research Notes

    Article Title: Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

    doi: 10.1186/s13104-015-1813-5

    Figure Lengend Snippet: RNA integrity values for material isolated from LCM samples, whole tissue slices and cell lines. RNA quality (RIN values) for LCM derived samples, whole tissue slices and cell lines isolated with the RNeasy or miRNeasy kits. Median RIN values are shown, whiskers indicate interquartile range

    Article Snippet: RNA extraction and measurement The following RNA extraction kits were used to isolate total RNA from LCM acquired material: RNeasy® Micro (Qiagen, Germany), miRNeasy Mini (Qiagen, Germany), Arcturus® Picopure® RNA isolation kit (Arcturus, Applied Biosystems, USA.), mirVana™ miRNA isolation kit (Ambion, USA.) and RNAqueous® -Micro (Ambion, USA.).

    Techniques: Isolation, Laser Capture Microdissection, Derivative Assay

    Comparison of RNA extraction kits for microdissected samples. RNA quality for benign and PCa LCM samples are shown for all RNA extraction kits. RIN values are shown as the average of a duplicate experiment. RNeasy and miRNeasy consistently delivered high quality RNA from LCM samples ( a ). Median RIN values were compared between the groups, which were expected to be similar. However, RNeasy generated higher quality RNA than Picopure and RNAqueous (p = 0.01 and 0.08 respectively). Additionally, RNA quality between benign glands and PCa cells within the same patient was variable ( b )

    Journal: BMC Research Notes

    Article Title: Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

    doi: 10.1186/s13104-015-1813-5

    Figure Lengend Snippet: Comparison of RNA extraction kits for microdissected samples. RNA quality for benign and PCa LCM samples are shown for all RNA extraction kits. RIN values are shown as the average of a duplicate experiment. RNeasy and miRNeasy consistently delivered high quality RNA from LCM samples ( a ). Median RIN values were compared between the groups, which were expected to be similar. However, RNeasy generated higher quality RNA than Picopure and RNAqueous (p = 0.01 and 0.08 respectively). Additionally, RNA quality between benign glands and PCa cells within the same patient was variable ( b )

    Article Snippet: RNA extraction and measurement The following RNA extraction kits were used to isolate total RNA from LCM acquired material: RNeasy® Micro (Qiagen, Germany), miRNeasy Mini (Qiagen, Germany), Arcturus® Picopure® RNA isolation kit (Arcturus, Applied Biosystems, USA.), mirVana™ miRNA isolation kit (Ambion, USA.) and RNAqueous® -Micro (Ambion, USA.).

    Techniques: RNA Extraction, Laser Capture Microdissection, Generated

    Validation of gene silencing in HEp-2C and Vero cells by quantitative PCR (qPCR). siRNAs targeting selected hit genes were transfected into HEp-2C or Vero cells. At 48 h posttransfection, cells were harvested for RNA isolation using a Qiagen RNeasy minikit.

    Journal: Journal of Virology

    Article Title: Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

    doi: 10.1128/JVI.01464-15

    Figure Lengend Snippet: Validation of gene silencing in HEp-2C and Vero cells by quantitative PCR (qPCR). siRNAs targeting selected hit genes were transfected into HEp-2C or Vero cells. At 48 h posttransfection, cells were harvested for RNA isolation using a Qiagen RNeasy minikit.

    Article Snippet: Forty-eight hours after siRNA transfection, total RNA was isolated from mock-infected and transfected HEp-2C cells and Vero cells using an RNeasy minikit (Qiagen) according to the manufacturer's protocol.

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Isolation

    Indel Formation in CD34+ HSPCs Nucleofected with Modified Cas9 mRNAs CD34+ HSPCs were nucleofected with 3 μg of the indicated Cas9 mRNA and 2 μg IL2RGlocus MS-sgRNA. 6 μg Cas9 RNP complexed to 3.2 μg IL2RGlocus MS-sgRNA was nucleofected for comparison. ARCA 5meC/Ψ is our previously published Cas9 mRNA 6 and was also included for comparison. Cells were isolated after 4 days, and indel formation was assessed by TIDE analysis. Bars represent mean ± SEM of at least 5 independent transfections. White and gray bars indicate RNeasy and HPLC-purified mRNAs, respectively. sgRNA complexed to Cas9 RNP was included as a control. ***p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification

    doi: 10.1016/j.omtn.2018.06.010

    Figure Lengend Snippet: Indel Formation in CD34+ HSPCs Nucleofected with Modified Cas9 mRNAs CD34+ HSPCs were nucleofected with 3 μg of the indicated Cas9 mRNA and 2 μg IL2RGlocus MS-sgRNA. 6 μg Cas9 RNP complexed to 3.2 μg IL2RGlocus MS-sgRNA was nucleofected for comparison. ARCA 5meC/Ψ is our previously published Cas9 mRNA 6 and was also included for comparison. Cells were isolated after 4 days, and indel formation was assessed by TIDE analysis. Bars represent mean ± SEM of at least 5 independent transfections. White and gray bars indicate RNeasy and HPLC-purified mRNAs, respectively. sgRNA complexed to Cas9 RNP was included as a control. ***p

    Article Snippet: We initially made anti-reverse CleanCap trimers with a 3′-O-methyl group on the sugar of the m7 G to prevent incorporation in the opposite orientation, but we found this to be unnecessary, as the 3′-O-methyl version functioned equivalently to CleanCap with a 3′ OH. mRNAs were purified by RNeasy Maxi (QIAGEN, 75162), phosphatase treated for 1 hr with final 0.25 U/μg Antarctic phosphatase (M0289L) in 1× Antarctic phosphatase buffer, and then re-purified by RNeasy.

    Techniques: Modification, Mass Spectrometry, Isolation, Transfection, High Performance Liquid Chromatography, Purification

    RNA extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Journal: Frontiers in Immunology

    Article Title: An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

    doi: 10.3389/fimmu.2020.00402

    Figure Lengend Snippet: RNA extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Article Snippet: Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration Next, RNeasy® Mini and Micro silica columns (both QIAGEN) and MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy® MiniElute Cleanup Kit (QIAGEN).

    Techniques: RNA Extraction, Concentration Assay, Purification, Quantitative RT-PCR, Expressing, Cell Culture, Isolation, Real-time Polymerase Chain Reaction

    Reverse transcription evaluation. Four reverse transcription kits were evalued for relative qPCR signal for (A) maximal RNA yield, or (B) maximal RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). RNA was reverse transcribed with either Superscript™ III (SSIII), Superscript™ IV (SSIV) (both Invitrogen), iScript™ Advanced (iScript) (BioRad) or High-Capacity (HC) (ThermoFisher) reverse transcription kits. RNA was extracted from 1 × 10 6 PBMCs pared samples, cultured with complete media (white) or PMA/Iono (gray) for 6 h, then IFN- γ, RPL13a, SDHA , and TBP mRNA expression was quantified. Data were compared with a two-way ANOVA (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 for post-hoc Bonferroni's multiple-comparisons test). Biological replicate ( n = 4), single RNA extractions, with triplicate reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the reverse transcription reactions calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Journal: Frontiers in Immunology

    Article Title: An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

    doi: 10.3389/fimmu.2020.00402

    Figure Lengend Snippet: Reverse transcription evaluation. Four reverse transcription kits were evalued for relative qPCR signal for (A) maximal RNA yield, or (B) maximal RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). RNA was reverse transcribed with either Superscript™ III (SSIII), Superscript™ IV (SSIV) (both Invitrogen), iScript™ Advanced (iScript) (BioRad) or High-Capacity (HC) (ThermoFisher) reverse transcription kits. RNA was extracted from 1 × 10 6 PBMCs pared samples, cultured with complete media (white) or PMA/Iono (gray) for 6 h, then IFN- γ, RPL13a, SDHA , and TBP mRNA expression was quantified. Data were compared with a two-way ANOVA (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 for post-hoc Bonferroni's multiple-comparisons test). Biological replicate ( n = 4), single RNA extractions, with triplicate reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the reverse transcription reactions calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Article Snippet: Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration Next, RNeasy® Mini and Micro silica columns (both QIAGEN) and MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy® MiniElute Cleanup Kit (QIAGEN).

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Isolation, Cell Culture, Expressing

    Assay analytical sensitivity. Relative RT-qPCR signal for IFN- γ, RPL13a, SDHA , and TBP mRNA expression from log 10 dilutions of unstimulated PBMCs when (A) maximizing RNA yield, or (B) maximizing RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ IV (Invitrogen). mRNA expression was determined by absolute-quantitative RT-qPCR and gene copy number per reaction was normalized to log 10 copies per reaction. Biological replicate ( n = 3), single RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Journal: Frontiers in Immunology

    Article Title: An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

    doi: 10.3389/fimmu.2020.00402

    Figure Lengend Snippet: Assay analytical sensitivity. Relative RT-qPCR signal for IFN- γ, RPL13a, SDHA , and TBP mRNA expression from log 10 dilutions of unstimulated PBMCs when (A) maximizing RNA yield, or (B) maximizing RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ IV (Invitrogen). mRNA expression was determined by absolute-quantitative RT-qPCR and gene copy number per reaction was normalized to log 10 copies per reaction. Biological replicate ( n = 3), single RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Article Snippet: Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration Next, RNeasy® Mini and Micro silica columns (both QIAGEN) and MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy® MiniElute Cleanup Kit (QIAGEN).

    Techniques: Quantitative RT-PCR, Expressing, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction

    qPCR optimization. (A) Experimental workflow for qPCR optimization. (B) Effect of stimulation on mRNA expression of reference genes RPL13a, SDHA , and TBP . 1 × 10 6 PBMCs paired samples were cultured with complete media (white), or stimulated with PMA/Iono control (gray) for 0, 6, 12, 16, 24, or 48 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, and reverse transcribed with Superscript™ III (Invitrogen). RNA expression was determined by absolute quantitative RT-qPCR wherein number of gene copies per reaction was quantified by standard curve and normalized to cell number. Data were compared with a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (** P ≤ 0.01; *** P ≤ 0.001). Biological replicate ( n = 3) single RNA extractions with single reverse transcription reactions per extraction were performed. Sample mean calculated from technical triplicate qPCR. Biological mean ± biological SEM are shown.

    Journal: Frontiers in Immunology

    Article Title: An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

    doi: 10.3389/fimmu.2020.00402

    Figure Lengend Snippet: qPCR optimization. (A) Experimental workflow for qPCR optimization. (B) Effect of stimulation on mRNA expression of reference genes RPL13a, SDHA , and TBP . 1 × 10 6 PBMCs paired samples were cultured with complete media (white), or stimulated with PMA/Iono control (gray) for 0, 6, 12, 16, 24, or 48 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, and reverse transcribed with Superscript™ III (Invitrogen). RNA expression was determined by absolute quantitative RT-qPCR wherein number of gene copies per reaction was quantified by standard curve and normalized to cell number. Data were compared with a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (** P ≤ 0.01; *** P ≤ 0.001). Biological replicate ( n = 3) single RNA extractions with single reverse transcription reactions per extraction were performed. Sample mean calculated from technical triplicate qPCR. Biological mean ± biological SEM are shown.

    Article Snippet: Magnetic Bead-Based Extraction Significantly Increased RNA Yield and Concentration Next, RNeasy® Mini and Micro silica columns (both QIAGEN) and MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy® MiniElute Cleanup Kit (QIAGEN).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, RNA Expression, Quantitative RT-PCR