RNP8 was assembled by incubating the dsRBD octamer and siRNA-DUPA in DPBS for 5 min under sterile condition. 2 × 105 LNCaP cells were treated with fresh RNP8 for 12 h in RPMI 1640 media plus 10% Q-serum42 (link), 54 (link), followed by a media exchange. As a positive control, cells were treated with 100 nM siRNA in the presence of Lipofectamine 2000 according to the manufacturer’s recommended protocol (Thermofisher, Waltham, MA). Thirty-two hours post-treatment, cells were harvested for RT-PCR and immunoblotting. Briefly, total RNA was isolated and reverse transcribed into cDNA using random hexamer and Taqman® Reverse Transcription reagents (Thermofisher, Waltham, MA). mRNA expression was measured using SensiFast SYBR kit (Bioline, Taunton, MA) on Chromo4™ Real-Time system (Bio-Rad, Hercules, CA). Plk1 mRNA expression level was normalized to peptidylprolyl isomerase A (PPIA) and expressed as the percentage of negative control. Immunoblotting was performed as previously reported55 (link) and all antibodies were purchased from ThermoFisher. The immunoblots were imaged on an Odyssey®CLx imaging system (Li-cor, Lincoln, NE) and analyzed with Image Studio Lite.
Chromo4 real time system
Manufactured by Bio-Rad
Sourced in United States
The Chromo4™ Real-Time system is a thermal cycler designed for real-time PCR (polymerase chain reaction) analysis. It is capable of detecting and quantifying nucleic acid sequences in real-time through the use of fluorescent dyes or probes. The system provides accurate and reliable data for a variety of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Sensifast sybr kit, by Meridian Bioscience (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green mix, by Qiagen (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Revertra ace kit, by Toyobo (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Pgem t easy vector system, by Promega (1 mentions)
Sybr green master mix, by Qiagen (1 mentions)
4 protocols using chromo4 real time system
1
RNP8-Mediated Silencing of Plk1 in LNCaP Cells
2
RNP8-Mediated Silencing of Plk1 in LNCaP Cells
3
Quantification of PNPLA2 and ALOX5 mRNA
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Total RNA was purified using RNeasy mini kits (Qiagen, Valencia, CA, USA), and 1 μg mRNA was reverse transcribed using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). PNPLA2 or ALOX5 mRNA levels were normalized to 18S levels by quantitative RT-PCR using SYBR Green mix (Qiagen) in the Bio-Rad Chromo4 real-time system (Hercules, CA, USA). The sequences of forward and reverse primers used are in the Table .
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was purified using the NucleoSpin RNA XS Purification Kit (Takara Bio, Inc., Shiga, Japan) and converted to cDNA using Moloney murine leukemia virus reverse transcriptase and random primers (ReverTra Ace kit; Toyobo, Tokyo, Japan). The QuantiTect SYBR Green PCR Kit (QIAGEN, Valencia, CA, USA) and the Chromo4 Real-Time System (Bio-Rad, Hercules, CA, USA) were used to perform qRT-PCR. Reaction mixtures (20 μL) for qRT-PCR consisted of 2 μL of cDNA, 10 μL of SYBR Green Master Mix (Qiagen GmbH, Hilden, Germany), and the appropriate primers. Primer sequences are shown in Table 1. Product synthesis was monitored by measuring SYBR Green fluorescence levels, which were subsequently normalized to the fluorescence levels associated with 18S ribosomal RNA. For all qRT-PCR analyses, we used absolute quantification by using standard plasmids. To obtain plasmids for use as standards for qRT-PCR, an amplified cDNA fragment of the gene of interest was cloned into the pGEM™-T Easy Vector System (Promega Corp., Madison, WI, USA).
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