Total RNA from the cells and exosomes was isolated using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) or E.Z.N.A.™ miRNA Kit (Omega Bio-tek, Norcross, GA, USA) in accordance with the manufacturer’s instructions. First-strand cDNA was synthesized from RNA primed by oligo (dT) using M-MLV reverse transcriptase. The RT-qPCR assay for multiple genes was performed with the miScript SYBR Green PCR Kit by an CFX96™ Real-Time PCR Dectection System (BIO-RAD, Hercules, CA, USA). The primer of miRNA sequences is listed in Table 1
Miscript sybr green pcr kit
Manufactured by Bio-Rad
Sourced in United States
The MiScript SYBR Green PCR Kit is a qPCR (quantitative PCR) reagent designed for the detection and quantification of microRNA (miRNA) expression. The kit includes all the necessary components for real-time PCR amplification and detection, including the SYBR Green dye for fluorescent signal generation.
Automatically generated - may contain errors
Lab products found in correlation
Miscript 2 rt kit, by Qiagen (4 mentions)
Quantifast sybr green rt pcr kit, by Qiagen (2 mentions)
Mirna and mrna primer assays, by Qiagen (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
E z n a mirna kit, by Omega Bio-Tek (1 mentions)
Icycler thermal cycler, by Bio-Rad (1 mentions)
Platinum taq polymerase, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Miscript cdna synthesis kit, by Bio-Rad (1 mentions)
Epicentre s pcr premix f, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Bio-Rad (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Centrifugal filter unit, by Merck Group (1 mentions)
Geneamp pcr system 9700, by Qiagen (1 mentions)
7 protocols using miscript sybr green pcr kit
1
Quantifying miRNA Expression in Cells and Exosomes
2
Quantitative Analysis of mRNA and miRNA Expression
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RNA was collected using TRIzol® according to standard protocol: https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf . RT-PCR was performed according to the QIAGEN provided protocol. QIAGEN SYBR® Green QuantiFast RT-PCR kit and protocol was utilized for quantification of mRNA, and QIAGEN miScript II RT Kit and protocol along with QIAGEN miScript SYBR® Green PCR Kit and protocol was utilized for quantification of miRNA with the Bio-Rad CFX Connect™ Real-Time PCR Detection System. miRNA and mRNA primer assays from QIAGEN were utilized. mRNA was normalized to GAPDH and miRNA was normalized to RNU6. Exosomal RNA was collected with the ExoQuick-TC™ reagent and TRIzol® RNA isolation method. Exosome pellets were incubated in TRIzol® for 1–2 hours prior to the isolation procedure. See exosome and RNA collection methods above.
3
OMV Modulates VSMC Marker Expression
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The effect of OMV on the expression of VSMC markers was determined by real‐time PCR. Total RNAs were isolated from VSMCs using Trizol reagent and reverse‐transcribed into cDNA. SYBR Green‐based real‐time PCR reactions were performed using specific primers for murine ALP 38 , ColIA1 16 , OC 17 , Runx2 20 , SM22α 21 , α‐SMA 21 ,ERK1/2 29 , and β‐actin as the control 38 . Quantitative real‐time PCR was performed with the miScript SYBR Green PCR Kit on an iCycler Thermal Cycler (Bio‐Rad, Hercules, CA, USA) according to the manufacturer's instructions.
4
Immune Modulation Protocol using miR-185-5p
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The following chemicals were purchased and used: SEB (Toxin Technologies, Sarasota, FL, United States), RES and DMSO (Sigma-Aldrich, St. Louis, MO, United States), culture medium (RPMI 1640), Penicillin/Streptomycin, HEPES, L -glutamine, FBS, and PBS (Invitrogen Life Technologies, Carlsbad, CA, United States). Fluorophore-labeled anti-mouse CD3, CD4, CD8, CD44, NK1.1, CD11b, and Gr1 antibodies were purchased from eBioScience (Carlsbad, CA, United States). Bio-Plex kit for mouse cytokines was purchased from Bio-Rad (Bio-Rad, Hercules, CA, United States). Polymerase chain reaction (PCR) reagents, Epicentre’s PCR premix F and Platinum Taq Polymerase, were purchased form Invitrogen Life Technologies (Carlsbad, CA, United States). miRNeasy kit, miScript cDNA synthesis kit, miScript primer assays kit, miScript SYBR Green PCR kit, miR-185-5P mimic, and miR-185-5p inhibitor, SsoAdvanced SYBR green supermix from Bio-Rad (Hercules, CA, United States) were purchased from QIAGEN (Qiagen, Inc., Valencia, CA, United States).
5
Extracellular miRNA Extraction and Quantification
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Cell culture media were collected after cell treatment and concentrated by Centrifugal Filter Units (Millipore). Rat blood samples were collected and centrifuged to obtain plasma. MiRNAs were extracted from cell culture media and from rat plasma by miRNeasy Serum/Plasma Kit (Qiagen) and from circulating exosomes by exoRNeasy Serum/Plasma Midi Kit(Qiagen) according to the manufacturer's instructions. RT were performed by miScript II RT kit (Qiagen) and GeneAmp PCR System 9700. qPCR were performed by using the miScript SybrGreen PCR Kit using the CFX96 Real-time system (Bio-Rad). qPCR used miScript Primer Assay. MiR-34a levels were detected and normalized by using C. elegans miR-39 miRNeasy Serum/Plasma Spike-in control. Relative expression was calculated using the comparative cycle threshold (Ct) method (2−ΔΔCt).
6
Quantitative Analysis of mRNA and miRNA Expression
7
Quantitative Analysis of Angiogenic Factors
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Total RNA was extracted from the tissue samples using mirVana™ miRNA isolation kit (Ambion-Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from RNA using miScript II RT kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Real-time PCR was performed using miScript SYBR-Green PCR kit in an automated thermal cycler (Bio-Rad Laboratories). miR-410 and SNORD68 primers were obtained from Qiagen. SNORD68 was used as an internal control for normalization. The expression levels of pro-angiogenic factors VEGF-A, -B, -C, FGF2, PDGF, ANGPT1 and ANGPT2 (Table I ) were measured as per standard protocol and normalized to HPRT. Relative gene expression was calculated by the 2−ΔCt method.
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