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Ovation picosl wta v2 kit

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The Ovation PicoSL WTA V2 kit is a lab equipment product designed for whole transcriptome amplification from low-input or degraded RNA samples. It provides a streamlined workflow for preparing amplified cDNA libraries from small amounts of input RNA.

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Lab products found in correlation

Rneasy plus micro kit, by Qiagen (4 mentions) Bioanalyzer, by Agilent Technologies (4 mentions) Facsaria, by BD (4 mentions) Rnaprotect cell reagent, by Qiagen (4 mentions) Taqman assay, by Thermo Fisher Scientific (4 mentions) Taqman probe, by Thermo Fisher Scientific (3 mentions) Abi rt pcr stepone plus system, by Thermo Fisher Scientific (3 mentions) Stepone, by Thermo Fisher Scientific (2 mentions) Influx cell sorter, by BD (2 mentions) Magnetic bead selection, by Miltenyi Biotec (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Realplex2, by Eppendorf (2 mentions) Rna reliprep kit, by Promega (2 mentions) Faststart universal sybr green master mix, by Roche (2 mentions) Influx high speed cell sorter, by BD (2 mentions) Stepone real time pcr system instrument, by Thermo Fisher Scientific (2 mentions) Abi steponeplus system, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Rnase mini kit, by Qiagen (1 mentions)

10 protocols using ovation picosl wta v2 kit

1

RNA Isolation and Quantification for FRCs

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For RNA processing, cell sorting was performed on a high-speed Influx Cell Sorter or a FACSAria (100 µmol/L nozzle, both BD Biosciences) into RNA protect Cell Reagent (QIAGEN, #76526). Lymph node FRCs cell suspensions were sorted based on expression of Pdpn (clone #8.1.1) within the CD45 (clone #30-F11), and CD31 (clone #MEC13.3; all BioLegend) negative gate. RNA was then isolated with the RNeasy plus micro Kit (QIAGEN, #74034) or the ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific, #KIT0204) and RNA quality and quantity was analyzed with a Bioanalyzer (Agilent Technologies). Only RNA samples with a RNA integrity number (RIN) above 8 and total concentration of 100 pg/µL were further processed for whole transcriptome amplification via the Ovation PicoSL WTA V2 Kit (NuGEN, #3312). qRT-PCR using 20 ng cDNA input material was performed using TaqMan assays with the following primers/probes (all Applied Biosystems): Pdpn Mm01348912_g1, Thy1 Mm00493682_g1, Il7 Mm01295803_m1, and housekeeping gene Actb Mm00607939_s1. qRT-PCR was performed on a StepOne or ViiA 7 Real Time PCR System instrument in a relative quantification setting (both Life Technologies). Gene expression level are shown as 2–ddCt.
Riedel A., Helal M., Pedro L., Swietlik J.J., Shorthouse D., Schmitz W., Haas L., Young T., da Costa A.S., Davidson S., Bhandare P., Wolf E., Hall B.A., Frezza C., Oskarsson T, & Shields J.D. (2022). Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma. Cancer Immunology Research, 10(4), 482-497.
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2

Quantifying NKAP and Rag1 mRNA in iNKT cells

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mRNA was isolated from sorted populations using an RNeasy kit (Qiagen). cDNA from sorted iNKT cells was generated and amplified using an Ovation PicoSL WTA V2 kit (NuGen). Taqman probes (Applied Biosystems) for NKAP, Rag1 and Gapdh were used. An ABI StepOne Plus System (Applied Biosystems) was used and relative expression was calculated with the 2-ΔΔCT method (31 ). The relative mRNA expression of NKAP or Rag1 from the sorted iNKT cells was analyzed using Q-PCR. Statistics were calculated comparing NKAP expression in PLZF-cre NKAP cKO to WT cells using unpaired student’s t test.
Thapa P., Chen M.W., McWilliams D.C., Belmonte P., Constans M., Sant’Angelo D.B, & Shapiro V.S. (2016). NKAP regulates iNKT cell proliferation and differentiation into ROR-γt expressing NKT17 cells. Journal of immunology (Baltimore, Md. : 1950), 196(12), 4987-4998.
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3

Isolation and Analysis of CD4+ T Cell Subsets

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To isolate CD4+ T cells, splenocytes from Rag1-GFP WT and Rag1-GFP CD4-cre HDAC3 cKO mice were harvested and negatively selected from biotin-labeled populations (the biotin-cocktail contained B220, CD11b, Ter119, Gr-1, CD19, CD11c) using magnetic bead selection (Miltenyi Biotec). WT splenocytes lacking the Rag1-GFP reporter was used to to define gates for Rag1-GFP+ (RTE) and Rag1-GFP− (MNT) populations. All sorting was done on a FACSAria (BD Bioscience). mRNA was later isolated from sorted CD4+ RTEs (CD44−CD62L+Rag1-GFP+), CD4+ MNTs (CD44−CD62L+Rag1-GFP−), and memory CD4+ T cells (CD44+CD62L−Rag1-GFP−), with an RNeasy Mini kit (QIAGEN). cDNA was generated and amplified with the Ovation PicoSL WTA V2 kit (NuGen). mRNA expression was analyzed using TaqMan probes (Applied Biosystems) for HDAC3, St8sia1, St8sia4, St8sia6, and 18S rRNA as an internal control. An ABI RT-PCR StepOne Plus System (Applied Biosystems) was used and, and gene expression calculated via the 2-ΔΔCT method.
Hsu F.C., Belmonte P., Constans M., Chen M.W., McWilliams D.C., Hiebert S.W, & Shapiro V.S. (2015). HDAC3 is required for T cell maturation. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1578-1590.
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4

Isolation and Analysis of Splenic CD4+ RTEs

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To isolate splenic CD4+ RTEs, cells from Rag1-GFP WT and Rag1-GFP CD4-cre Runx1 cKO mice were harvested and first subject to negative selection using a biotin-labeled cocktail of antibodies (B220, CD11b, Ter119, Gr-1, CD19, CD11c and CD8) to eliminate unwanted populations using magnetic bead selection (Miltenyi Biotec) prior to sorting. All sorting was done on a FACSAria (BD Bioscience), and RTEs were sorted by gating on CD4+ CD44−CD62L+ Rag1-GFP+. mRNA was later isolated from sorted CD4+ RTEs using an RNeasy Mini kit (QIAGEN). cDNA was then generated and amplified with the Ovation PicoSL WTA V2 kit (NuGen). The mRNA expression was measured using TaqMan probes (Applied Biosystems) for ST8Sia1, ST8Sia4, ST8Sia6, ST3Gal1, ST3Gal2 and ST3Gal6. TaqMan probe for 18S rRNA is used as the internal control. Samples were analyzed using an ABI RT-PCR StepOne Plus System (Applied Biosystems), and relative gene expression was calculated via the 2-ΔΔCT method. Data shown is from independent isolations of CD4+ RTEs from 3 Rag1-GFP WT and 3 Rag1-GFP CD4-cre Runx1 cKO mice.
Hsu F.C., Shapiro M.J., Dash B., Chen C.C., Constans M.M., Chung J.Y., Romero Arocha S.R., Belmonte P.J., Chen M.W., McWilliams D.C, & Shapiro V.S. (2016). An Essential Role for the Transcription Factor Runx1 in T Cell Maturation. Scientific Reports, 6, 23533.
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5

RNA Isolation and RT-PCR Analysis

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RNA was isolated from sorted cells using the Promega RNA Reliprep kit. cDNA was synthesized and amplified using the Ovation PicoSL WTA V2 kit (NuGen). RT-PCR reactions were performed using Faststart Universal SYBR green Master Mix (Roche) and run on an Eppendorf Realplex2 (link) thermocycler. Primer sequences are as listed:
Vaughan A.E., Brumwell A.N., Xi Y., Gotts J., Brownfield D.G., Treutlein B., Tan K., Tan V., Liu F., Looney M.R., Matthay M., Rock J.R, & Chapman H.A. (2014). Lineage-negative Progenitors Mobilize to Regenerate Lung Epithelium after Major Injury. Nature, 517(7536), 621-625.
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6

Quantification of HDAC3 Expression in iNKT Cells

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cDNA was generated and amplified using an Ovation PicoSL WTA V2 kit (NuGen) from mRNA (Qiagen RNase mini kit) isolated from sorted iNKT cells. Taqman Hdac3 and Gapdh gene expression assays were purchased from Applied Biosystems. The Taqman assay Hdac3 spanned a deleted exon (exon 7) in the Hdac3 gene. Relative expression of Hdac3 was calculated using 2−ΔΔCT method54 (link), q-PCR was performed in ABI RT-PCR StepOne Plus System (Applied Biosystems). Hdac3 expression was calculated relative to WT Stage 3 (=1).
Thapa P., Romero Arocha S., Chung J.Y., Sant’Angelo D.B, & Shapiro V.S. (2017). Histone deacetylase 3 is required for iNKT cell development. Scientific Reports, 7, 5784.
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7

Quantitative RNA Analysis of Lymph Node Cells

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For RNA processing, LNs cell suspensions were sorted on a High speed Influx Cell Sorter (100 μm nozzle, BD Biosciences) into RNA protect Cell Reagent (QIAGEN). RNA was isolated with the RNeasy plus micro Kit (QIAGEN) and RNA quality and quantity was analyzed with a Bioanalyzer (Agilent Technologies). Only RNA samples with a RIN value above 8 and total concentration of 100 pg/μl were further processed for whole transcriptome amplification via the Ovation PicoSL WTA V2 Kit (NuGEN). qRT-PCR was performed using TaqMan assays (Pdpn Mm01348912_g1, Il7 Mm01295803_m1, Ccl21a Mm03646971_gH, S100a4 (FSP1) Mm00803372_g1, Thy1 Mm00493682_g1, Aqp1 Mm00431834_m1, Tyrp1 Mm00453201_m1, Dct Mm01225584_m1) and a StepOne Real Time PCR System instrument (both Life Technologies).
Riedel A., Shorthouse D., Haas L., Hall B.A, & Shields J. (2016). Tumor Induced Stromal Reprogramming Drives Lymph Node Transformation. Nature immunology, 17(9), 1118-1127.
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8

RNA Isolation and qRT-PCR Analysis of LN FRCs

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For RNA processing, cell sorting was performed on a High speed Influx Cell Sorter or a FACSAria (100 μm nozzle, both BD Biosciences) into RNA protect Cell Reagent (QIAGEN, #76526). LN FRCs cell suspensions were sorted based on expression of Pdpn (clone #8.1.1) within the CD45 (clone #30-F11), and CD31 (clone #MEC13.3) (all BioLegend) negative gate. RNA was then isolated with the RNeasy plus micro Kit (QIAGEN, #74034) or the ARCTURUS PicoPure RNA Isolation Kit (Thermo Fisher Scientific, #KIT0204) and RNA quality and quantity was analyzed with a Bioanalyzer (Agilent Technologies). Only RNA samples with a RNA integrity number (RIN) above 8 and total concentration of 100 pg/μl were further processed for whole transcriptome amplification via the Ovation PicoSL WTA V2 Kit (NuGEN, #3312). Quantitative reverse-transcription-PCR (qRT-PCR) using 20ng cDNA input material was performed using TaqMan assays with the following primers/probes (all Applied Biosystems): Pdpn Mm01348912_g1, Thy1 Mm00493682_g1, Il7 Mm01295803_m1, and housekeeping gene Actb Mm00607939_s1. qRT-PCR was performed on a StepOne or ViiA 7 Real Time PCR System instrument in a relative quantification setting (both Life Technologies). Gene expression level are shown as 2-ddCt.
Riedel A., Helal M., Pedro L., Swietlik J.J., Shorthouse D., Schmitz W., Haas L., Young T., da Costa A.S., Davidson S., Bhandare P., Wolf E., Hall B.A., Frezza C., Oskarsson T, & Shields J.D. (2022). Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma. Cancer Immunology Research, 10(4), 482-497.
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9

RNA Isolation and RT-PCR Analysis

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RNA was isolated from sorted cells using the Promega RNA Reliprep kit. cDNA was synthesized and amplified using the Ovation PicoSL WTA V2 kit (NuGen). RT-PCR reactions were performed using Faststart Universal SYBR green Master Mix (Roche) and run on an Eppendorf Realplex2 (link) thermocycler. Primer sequences are as listed:
Vaughan A.E., Brumwell A.N., Xi Y., Gotts J., Brownfield D.G., Treutlein B., Tan K., Tan V., Liu F., Looney M.R., Matthay M., Rock J.R, & Chapman H.A. (2014). Lineage-negative Progenitors Mobilize to Regenerate Lung Epithelium after Major Injury. Nature, 517(7536), 621-625.
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10

Quantitative RNA Analysis of Lymph Node Cells

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For RNA processing, LNs cell suspensions were sorted on a High speed Influx Cell Sorter (100 μm nozzle, BD Biosciences) into RNA protect Cell Reagent (QIAGEN). RNA was isolated with the RNeasy plus micro Kit (QIAGEN) and RNA quality and quantity was analyzed with a Bioanalyzer (Agilent Technologies). Only RNA samples with a RIN value above 8 and total concentration of 100 pg/μl were further processed for whole transcriptome amplification via the Ovation PicoSL WTA V2 Kit (NuGEN). qRT-PCR was performed using TaqMan assays (Pdpn Mm01348912_g1, Il7 Mm01295803_m1, Ccl21a Mm03646971_gH, S100a4 (FSP1) Mm00803372_g1, Thy1 Mm00493682_g1, Aqp1 Mm00431834_m1, Tyrp1 Mm00453201_m1, Dct Mm01225584_m1) and a StepOne Real Time PCR System instrument (both Life Technologies).
Riedel A., Shorthouse D., Haas L., Hall B.A, & Shields J. (2016). Tumor Induced Stromal Reprogramming Drives Lymph Node Transformation. Nature immunology, 17(9), 1118-1127.
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