rneasy plus mini kit Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen rneasy kit
    eIF3f induced rRNA degradation in pancreatic cells. (A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total <t>RNA</t> was isolated using <t>RNeasy</t> Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.
    Rneasy Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 117069 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy kit/product/Qiagen
    Average 99 stars, based on 117069 article reviews
    Price from $9.99 to $1999.99
    rneasy kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Qiagen rneasy extraction kit
    The decreased levels of <t>CCR5</t> expression by CCR5 shRNA (1005) correlates with that of CCR5 mRNA . huCCR5-293T cells were transduced with lentiviral vectors bearing either shRNA 1005 against CCR5 {CCR5shRNA (1005)} or a control shRNA against firefly luciferase (Luc shRNA). To monitor the expression levels of CCR5 on cell surface, the cells were cultured for 4 days and stained with either PE-Cy5 conjugated anti human CCR5 monoclonal antibody or isotype control. CCR5 and EGFP expression were analyzed by flow cytometry. The percentage number in each quadrant is indicated in each panel (A). To measure the levels of CCR5 mRNA, total RNA was isolated using Qiagen <t>RNeasy</t> extraction kit. Quantitative RT-PCR was performed using IQ5 with iScript one step RT-PCR kit using β-actin as an internal control (B).
    Rneasy Extraction Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3921 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy extraction kit/product/Qiagen
    Average 99 stars, based on 3921 article reviews
    Price from $9.99 to $1999.99
    rneasy extraction kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Qiagen rneasy mini plus kit
    The decreased levels of <t>CCR5</t> expression by CCR5 shRNA (1005) correlates with that of CCR5 mRNA . huCCR5-293T cells were transduced with lentiviral vectors bearing either shRNA 1005 against CCR5 {CCR5shRNA (1005)} or a control shRNA against firefly luciferase (Luc shRNA). To monitor the expression levels of CCR5 on cell surface, the cells were cultured for 4 days and stained with either PE-Cy5 conjugated anti human CCR5 monoclonal antibody or isotype control. CCR5 and EGFP expression were analyzed by flow cytometry. The percentage number in each quadrant is indicated in each panel (A). To measure the levels of CCR5 mRNA, total RNA was isolated using Qiagen <t>RNeasy</t> extraction kit. Quantitative RT-PCR was performed using IQ5 with iScript one step RT-PCR kit using β-actin as an internal control (B).
    Rneasy Mini Plus Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy mini plus kit/product/Qiagen
    Average 99 stars, based on 1666 article reviews
    Price from $9.99 to $1999.99
    rneasy mini plus kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    eIF3f induced rRNA degradation in pancreatic cells. (A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total RNA was isolated using RNeasy Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.

    Journal: PLoS ONE

    Article Title: The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation

    doi: 10.1371/journal.pone.0034194

    Figure Lengend Snippet: eIF3f induced rRNA degradation in pancreatic cells. (A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total RNA was isolated using RNeasy Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.

    Article Snippet: Cells were harvested at 4, 8 and 16 h after transfection and total RNA was isolated using RNeasy kit (QIAGEN). rRNAs (2.0 µg) were separated on an agarose gel and visualized by UV light (B).

    Techniques: Expressing, Transfection, Quantitative RT-PCR, Isolation, MTT Assay, Blocking Assay

    Representative gel electrophoresis of PCR reactions using universal bacterial primers and sputum RNA isolated with the RNeasy Plus Mini kit. Sputum cells were either subjected or not to bead vortexing (bashing or glass beads) prior to RNA isolation. Bands representing contaminating bacterial DNA are indicated. M: molecular weight marker.

    Journal: PLoS ONE

    Article Title: Elimination of bacterial DNA during RNA isolation from sputum: Bashing bead vortexing is preferable over prolonged DNase treatment

    doi: 10.1371/journal.pone.0214609

    Figure Lengend Snippet: Representative gel electrophoresis of PCR reactions using universal bacterial primers and sputum RNA isolated with the RNeasy Plus Mini kit. Sputum cells were either subjected or not to bead vortexing (bashing or glass beads) prior to RNA isolation. Bands representing contaminating bacterial DNA are indicated. M: molecular weight marker.

    Article Snippet: Optimizing RNA isolation using the RNeasy Plus Mini kit The RNeasy Plus Mini kit offers two alternative protocols; the standard protocol provides enrichment of the sample with intact mRNAs by eliminating RNAs shorter than 200 nucleotides; the second protocol is designed for the purification of total RNA.

    Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Isolation, Molecular Weight, Marker

    Cycle threshold (Ct) values for real-time PCR assays with different base pair (bp) long amplicons of glyceraldehyde 3-phophate dehydrogenase on RNA isolated from sputum samples. Samples were subjected either to repeated digestion process (up to 6 times) with the Turbo DNA-free kit or bead vortexing prior to RNA isolation using the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column (n = 6 for each group). Error bars indicate SEM. # p

    Journal: PLoS ONE

    Article Title: Elimination of bacterial DNA during RNA isolation from sputum: Bashing bead vortexing is preferable over prolonged DNase treatment

    doi: 10.1371/journal.pone.0214609

    Figure Lengend Snippet: Cycle threshold (Ct) values for real-time PCR assays with different base pair (bp) long amplicons of glyceraldehyde 3-phophate dehydrogenase on RNA isolated from sputum samples. Samples were subjected either to repeated digestion process (up to 6 times) with the Turbo DNA-free kit or bead vortexing prior to RNA isolation using the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column (n = 6 for each group). Error bars indicate SEM. # p

    Article Snippet: Optimizing RNA isolation using the RNeasy Plus Mini kit The RNeasy Plus Mini kit offers two alternative protocols; the standard protocol provides enrichment of the sample with intact mRNAs by eliminating RNAs shorter than 200 nucleotides; the second protocol is designed for the purification of total RNA.

    Techniques: Real-time Polymerase Chain Reaction, Isolation

    SPP1 expression is up-regulated in glioma initiating cells ( A ) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 10 7 ) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. ( B ) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and * *P ≤ 0.01. ( C ) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.

    Journal: Oncotarget

    Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma

    doi: 10.18632/oncotarget.14092

    Figure Lengend Snippet: SPP1 expression is up-regulated in glioma initiating cells ( A ) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 10 7 ) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. ( B ) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and * *P ≤ 0.01. ( C ) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.

    Article Snippet: Real- time PCR Total RNA was isolated from glioma cells using Qiagen RNeasy kit, 1 μg was used as a template.

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, Positive Control, FACS, Isolation, Real-time Polymerase Chain Reaction

    Replication Factors Are Misregulated in set2 Δ Cells (A) Transcript levels of cdc18 + , cdc22 + , and cdt1 + were established in exponentially growing wild-type and set2 Δ cells. RNA was extracted with Qiagen RNeasy kit and relative transcript levels of cdc18 + , cdc22 + , and cdt1 + were established by RT-qPCR. Error bars represent SD of three biological repeats. The asterisk ( ∗ ) represents significant difference compared with wild-type and set2 Δ. (B) Cdc18, Cdt1, or Cdc22 protein levels of exponentially growing wild-type and set2 Δ cells. Cell extracts were prepared from vegetative cells and processed for western blot. Immunoblots of total cell lysates were probed with PAP or GFP antibody. α-Tubulin is shown as a loading control. (C) cdc18-TAP or cdc18-TAP set2 Δ cells were arrested in G1 by nitrogen starvation and released, and samples were taken at time points indicated and subjected to FACS analysis. (D) In parallel, cdc18-TAP and cdc18-TAP set2 Δ cells were processed for western blotting at indicated times. (E) cdt1-TAP or cdt1-TAP set2 Δ cells were arrested in G1 by nitrogen starvation, released, and samples taken at time points indicated and subjected to FACS analysis. (F) In parallel, cdt1-TAP and cdt1-TAP set2 Δ cells were processed for western blotting at indicated times. (G) A similar experiment to that described in (C) was carried out using cdc22-CFP or cdc22-CFP set2 Δ cells. (H) A similar experiment to that described in (D) was carried out using cdc22-CFP or cdc22-CFP set2 Δ cells. Immunoblots of total cell extracts were probed with GFP antibody. α-Tubulin is shown as a loading control.

    Journal: Cell Reports

    Article Title: Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription

    doi: 10.1016/j.celrep.2017.08.058

    Figure Lengend Snippet: Replication Factors Are Misregulated in set2 Δ Cells (A) Transcript levels of cdc18 + , cdc22 + , and cdt1 + were established in exponentially growing wild-type and set2 Δ cells. RNA was extracted with Qiagen RNeasy kit and relative transcript levels of cdc18 + , cdc22 + , and cdt1 + were established by RT-qPCR. Error bars represent SD of three biological repeats. The asterisk ( ∗ ) represents significant difference compared with wild-type and set2 Δ. (B) Cdc18, Cdt1, or Cdc22 protein levels of exponentially growing wild-type and set2 Δ cells. Cell extracts were prepared from vegetative cells and processed for western blot. Immunoblots of total cell lysates were probed with PAP or GFP antibody. α-Tubulin is shown as a loading control. (C) cdc18-TAP or cdc18-TAP set2 Δ cells were arrested in G1 by nitrogen starvation and released, and samples were taken at time points indicated and subjected to FACS analysis. (D) In parallel, cdc18-TAP and cdc18-TAP set2 Δ cells were processed for western blotting at indicated times. (E) cdt1-TAP or cdt1-TAP set2 Δ cells were arrested in G1 by nitrogen starvation, released, and samples taken at time points indicated and subjected to FACS analysis. (F) In parallel, cdt1-TAP and cdt1-TAP set2 Δ cells were processed for western blotting at indicated times. (G) A similar experiment to that described in (C) was carried out using cdc22-CFP or cdc22-CFP set2 Δ cells. (H) A similar experiment to that described in (D) was carried out using cdc22-CFP or cdc22-CFP set2 Δ cells. Immunoblots of total cell extracts were probed with GFP antibody. α-Tubulin is shown as a loading control.

    Article Snippet: Total RNA was prepared using the RNeasy Plus Kit (Qiagen) as indicated in the manufacturer’s manual.

    Techniques: Quantitative RT-PCR, Western Blot, FACS

    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

    Induction of cytokine genes by POVPC and its metabolites. Time-dependent changes in the expression of (A) TNF-α, (B) IL-8, (C) GM-CSF, (D) IL-1β, (E) MCP-1, and (F) IL-6 in THP-DM treated with 10 μM each of POVPC, lyso-PC, and PHVPC. (G) Changes in cytokine gene expression in THP-DM treated with 10 μM of POVPE or PHVPE for 3 h. The THP-1 cells were differentiated with 100 nM PMA for 72 h, incubated in media with 0.1% FBS for an additional 24 h, and then either left untreated or treated in HBSS with POVPC or POVPE and their metabolites. At indicated times, the cells were harvested, and their total RNA was extracted with RNeasy® Mini Kit (Qiagen). Changes in the levels of mRNA of cytokine genes were measured by quantitative RT-PCR as described in Materials and Methods . Data are mean ± SEM; n = 3. * P

    Journal: Journal of Lipid Research

    Article Title: Reductive metabolism increases the proinflammatory activity of aldehyde phospholipids [S]

    doi: 10.1194/jlr.M013854

    Figure Lengend Snippet: Induction of cytokine genes by POVPC and its metabolites. Time-dependent changes in the expression of (A) TNF-α, (B) IL-8, (C) GM-CSF, (D) IL-1β, (E) MCP-1, and (F) IL-6 in THP-DM treated with 10 μM each of POVPC, lyso-PC, and PHVPC. (G) Changes in cytokine gene expression in THP-DM treated with 10 μM of POVPE or PHVPE for 3 h. The THP-1 cells were differentiated with 100 nM PMA for 72 h, incubated in media with 0.1% FBS for an additional 24 h, and then either left untreated or treated in HBSS with POVPC or POVPE and their metabolites. At indicated times, the cells were harvested, and their total RNA was extracted with RNeasy® Mini Kit (Qiagen). Changes in the levels of mRNA of cytokine genes were measured by quantitative RT-PCR as described in Materials and Methods . Data are mean ± SEM; n = 3. * P

    Article Snippet: RNeasy RNA isolation kit was from Qiagen (Valencia, CA), rDNase (DNA-freeTM Kit) was from Ambion (Austin, TX), AMV Reverse Transcriptase from Promega (Madison, WI), and SYBR Green master mix from Quanta BioSciences (Gaithersburg, MD).

    Techniques: Expressing, Incubation, Quantitative RT-PCR

    Estrogen induces VEGF mRNA production and protein secretion. A . 201T cells were serum starved for 48 h and then treated with 10 nM β-estradiol from 0–24 h. mRNA was isolated using RNeasy purification kit and RT-PCR was performed using

    Journal: Journal of Thoracic Oncology

    Article Title: Combining the Multi-Targeted Tyrosine Kinase Inhibitor Vandetanib with the Anti-Estrogen Fulvestrant Enhances its Anti-tumor Effect in Non-Small Cell Lung Cancer

    doi: 10.1097/JTO.0b013e31824177ea

    Figure Lengend Snippet: Estrogen induces VEGF mRNA production and protein secretion. A . 201T cells were serum starved for 48 h and then treated with 10 nM β-estradiol from 0–24 h. mRNA was isolated using RNeasy purification kit and RT-PCR was performed using

    Article Snippet: Cells were serum starved for 48 h and then treated with 10 nM β-estradiol from 0–24 h. In , cells were pretreated with inhibitors as indicated followed by treatment with 10 nM β-estradiol for 1 h. mRNA was isolated using RNeasy purification kit (Qiagen, Valencia, CA) and RT-PCR was performed using OneStep RT-PCR kit (Qiagen) with human VEGFA primer pair (BioChain Institute, Inc., Hayward, CA) and GAPDH as a control.

    Techniques: Isolation, Purification, Reverse Transcription Polymerase Chain Reaction

    RT-PCR analysis of APRT and HPRT mRNA. (A) Total RNA was extracted from CHO cell lines carrying a deletion of the APRT gene, the wild-type (wt) APRT, and APRT genes with insertions of CAG repeats. (B) Total RNA was extracted from CHO cells transiently transfected with the control plasmid, a plasmid containing the intact human HPRT minigene, and a set of plasmids containing CAG repeat tracts of various lengths cloned into the intron of the HPRT minigene. Junctions between exons 2 and 3 of the hamster APRT gene and junctions between exons 2 and 3 of the human HPRT minigene were amplified by RT-PCR and were separated on agarose gels. The lower band in each lane corresponds to the product expected for correct splicing of exons 2 and 3. Upper bands correspond to transcripts containing the CAG exon incorporated between exons 2 and 3. MW, molecular weight.

    Journal: Molecular and Cellular Biology

    Article Title: Selectable System for Monitoring the Instability of CTG/CAG Triplet Repeats in Mammalian Cells

    doi: 10.1128/MCB.23.13.4485-4493.2003

    Figure Lengend Snippet: RT-PCR analysis of APRT and HPRT mRNA. (A) Total RNA was extracted from CHO cell lines carrying a deletion of the APRT gene, the wild-type (wt) APRT, and APRT genes with insertions of CAG repeats. (B) Total RNA was extracted from CHO cells transiently transfected with the control plasmid, a plasmid containing the intact human HPRT minigene, and a set of plasmids containing CAG repeat tracts of various lengths cloned into the intron of the HPRT minigene. Junctions between exons 2 and 3 of the hamster APRT gene and junctions between exons 2 and 3 of the human HPRT minigene were amplified by RT-PCR and were separated on agarose gels. The lower band in each lane corresponds to the product expected for correct splicing of exons 2 and 3. Upper bands correspond to transcripts containing the CAG exon incorporated between exons 2 and 3. MW, molecular weight.

    Article Snippet: For RT-PCR, total RNA was extracted from cells by using an RNA-Easy kit (Qiagen) and was then reverse transcribed and amplified with a Titan Single Tube RT-PCR kit (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Clone Assay, Amplification, Molecular Weight

    The decreased levels of CCR5 expression by CCR5 shRNA (1005) correlates with that of CCR5 mRNA . huCCR5-293T cells were transduced with lentiviral vectors bearing either shRNA 1005 against CCR5 {CCR5shRNA (1005)} or a control shRNA against firefly luciferase (Luc shRNA). To monitor the expression levels of CCR5 on cell surface, the cells were cultured for 4 days and stained with either PE-Cy5 conjugated anti human CCR5 monoclonal antibody or isotype control. CCR5 and EGFP expression were analyzed by flow cytometry. The percentage number in each quadrant is indicated in each panel (A). To measure the levels of CCR5 mRNA, total RNA was isolated using Qiagen RNeasy extraction kit. Quantitative RT-PCR was performed using IQ5 with iScript one step RT-PCR kit using β-actin as an internal control (B).

    Journal: Genetic Vaccines and Therapy

    Article Title: Characterization of a potent non-cytotoxic shRNA directed to the HIV-1 co-receptor CCR5

    doi: 10.1186/1479-0556-7-8

    Figure Lengend Snippet: The decreased levels of CCR5 expression by CCR5 shRNA (1005) correlates with that of CCR5 mRNA . huCCR5-293T cells were transduced with lentiviral vectors bearing either shRNA 1005 against CCR5 {CCR5shRNA (1005)} or a control shRNA against firefly luciferase (Luc shRNA). To monitor the expression levels of CCR5 on cell surface, the cells were cultured for 4 days and stained with either PE-Cy5 conjugated anti human CCR5 monoclonal antibody or isotype control. CCR5 and EGFP expression were analyzed by flow cytometry. The percentage number in each quadrant is indicated in each panel (A). To measure the levels of CCR5 mRNA, total RNA was isolated using Qiagen RNeasy extraction kit. Quantitative RT-PCR was performed using IQ5 with iScript one step RT-PCR kit using β-actin as an internal control (B).

    Article Snippet: Quantitative RT-PCR for CCR5 mRNA Total RNAs from the infected CEM NKR-CCR5 cells were isolated using the Qiagen RNeasy extraction kit following manufacture's instruction.

    Techniques: Expressing, shRNA, Transduction, Luciferase, Cell Culture, Staining, Flow Cytometry, Cytometry, Isolation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

    Both yield and quality are variable within and across kit based methods, yet the modified CTAB protocol produces consistent high yield and quality in stored ‘d’Anjou tissues. a RINs are higher and more consistent across methods for stored ‘d’Anjou’ peel than cortex. b Excluding protocols with degraded RNA, yields are variable across kits with the highest yield using the CTAB protocol. c Excluding protocols with degraded RNA, A 260/280− ratios were also variable across methods, with CTAB again producing the cleanest RNA. Error bars are standard error of the mean, where applicable. Some data are missing due to very low yield or severely degraded individual samples. QRP RLC Qiagen RNeasy Plant using buffer RLC, CTAB our modified CTAB protocol see Additional file 1 , OHP Omega EZNA HP total RNA, TF thermo fisher, MN RAP Macherey–Nagel NucleoSpin Plant using buffer RAP, OTR Omega EZNA total RNA, QRP RLT Qiagen RNeasy Plant using buffer RLT, MN RA1 Macherey–Nagel NucleoSpin Plant using buffer RA1, ZR ZR plant RNA MiniPrep, OPR Omega EZNA plant RNA Kit 1, QRU Qiagen RNeasy plus universal

    Journal: BMC Research Notes

    Article Title: A practical examination of RNA isolation methods for European pear (Pyrus communis)

    doi: 10.1186/s13104-017-2564-2

    Figure Lengend Snippet: Both yield and quality are variable within and across kit based methods, yet the modified CTAB protocol produces consistent high yield and quality in stored ‘d’Anjou tissues. a RINs are higher and more consistent across methods for stored ‘d’Anjou’ peel than cortex. b Excluding protocols with degraded RNA, yields are variable across kits with the highest yield using the CTAB protocol. c Excluding protocols with degraded RNA, A 260/280− ratios were also variable across methods, with CTAB again producing the cleanest RNA. Error bars are standard error of the mean, where applicable. Some data are missing due to very low yield or severely degraded individual samples. QRP RLC Qiagen RNeasy Plant using buffer RLC, CTAB our modified CTAB protocol see Additional file 1 , OHP Omega EZNA HP total RNA, TF thermo fisher, MN RAP Macherey–Nagel NucleoSpin Plant using buffer RAP, OTR Omega EZNA total RNA, QRP RLT Qiagen RNeasy Plant using buffer RLT, MN RA1 Macherey–Nagel NucleoSpin Plant using buffer RA1, ZR ZR plant RNA MiniPrep, OPR Omega EZNA plant RNA Kit 1, QRU Qiagen RNeasy plus universal

    Article Snippet: While we did replace the reducing agent B-ME with the less toxic DTT, verified as a suitable B-ME replacement by Mommaerts et al. [ ], the CTAB method still included more hazardous material compared to most kits tested, excluding the Qiagen RNeasy Plus Universal kit which includes the QiaZol reagent and an organic extraction.

    Techniques: Modification