Allelic discrimination of HOTAIR rs920778 (assay ID: C_9162435_20), rs1899663 (assay ID: C_2104251_20), rs4759314 (assay ID: C_27930754_10), and rs12427129 (assay ID: C_2104247_10) SNPs was assessed using the TaqMan SNP Genotyping Assay with an ABI StepOnePlus™ Real-Time Polymerase Chain Reaction (PCR) System and further evaluated with SDS version 3.0 software (Applied Biosystems, Foster City, CA, USA) as described previously [31 (link)].
Steponeplus real time polymerase chain reaction
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOnePlus™ Real-Time Polymerase Chain Reaction (PCR) system is a laboratory instrument used for genetic analysis. It performs real-time PCR, which is a technique that allows for the simultaneous amplification and detection of specific DNA sequences. The system provides accurate quantification of target DNA or RNA molecules.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sds version 3, by Thermo Fisher Scientific (1 mentions)
Quantitect probe pcr master mix, by Qiagen (1 mentions)
Taqman probe based technology, by Thermo Fisher Scientific (1 mentions)
Primer perfect probe mix, by Qiagen (1 mentions)
Taqman probe mix, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Hiscript 2, by Vazyme (1 mentions)
Taqman array 96 well fast plate, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using steponeplus real time polymerase chain reaction
1
Allelic Discrimination of HOTAIR SNPs
2
Quantitative PCR Analysis of Inflammatory Markers
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Relative tumour necrosis factor-α (TNF-α), nuclear factor kappa beta (NFK-ß), and interleukin-6 (IL-6) messenger RNA (mRNA) expression analyses were performed using StepOne Plus Real-Time polymerase chain reaction (PCR) system technology (Applied Biosystems, Foster City, CA, USA) using synthesised cDNA from rat kidney RNA. A quantitative PCR was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using primers generated for rat TNF-α Rn00562055_m1, rat NFK-ß Rn01399583_m1, rat IL-6 Rn01410330_m1, and rat β-actin Rn00667869_m1. The results are expressed as relative-fold, compared with control animals. The expression data for β-actin in each tissue were used as the endogenous control. Each determination was performed in triplicate for each tissue in a 96-well optical plate for both targets, using 9-μL cDNA (100 ng), 1 μL of Primer Perfect Probe mix, and 10 μL of QuantiTect Probe PCR Master Mix (Qiagen) in each 20-μL reaction. The plates were heated for 2 min at 50 °C and then 10 min at 95 °C. Subsequently, 40 cycles of 15 s at 94 °C and cycles of 60 s at 60 °C were conducted. All data are expressed as the fold-change in expression compared with the expression in other animal groups, using the 2-delta-delta Ct (2-ΔΔCt) method [13 (link), 14 (link)].
3
Quantifying TINAG mRNA Expression
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Following the protocol of the manufacturer of the Trizol reagent (Invitrogen, USA), total RNA was extracted from the cells and tissues. The mRNA levels were determined using a LightCycler 480 (Roche, USA) or an Applied Biosystems StepOne Plus Real-Time Polymerase Chain Reaction machine to conduct the qRT-PCR analysis. Complementary deoxyribonucleic acid was synthesized using HiScript II (Vazyme, China). For the mRNA detection, glyceraldehyde 3-phosphate dehydrogenase served as the internal standard control. All the experiments were triplicated, and the results were compared using the Ct (cycle threshold) values. All the PCR primers were supplied by GeneCopoeia (Guangzhou, China). The sequences of tubulointerstitial nephritis antigen (TINAG) primers were F-CAGGTTCCAAGGAGAAGCCC and R-CAGGTTCCAAGGAGAAGCCC.
4
Gene Expression in Silicone Spacer Capsules
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RNA was extracted from the recipient subcutaneous fibrous capsules surrounding the silicone spacer at 6 weeks after pretreatment32
(AG; n = 5, GHNF; n = 5). The relative gene expression was determined using a TaqMan array 96-well FAST plate (4413257, Applied Biosystems; Bedford, Massachusetts, US). A TaqMan array plate contains 46 target genes and two assays for candidate endogenous control genes (Table 1 ). Samples were analyzed using a StepOnePlus Real-Time polymerase chain reaction (PCR) System (Applied Biosystems). The results were analyzed using ExpressionSuite Software ver. 1.3 (Applied Biosystems). The relative quantification (RQ) was calculated using the comparative computed tomography (CT) method. To determine the relative gene expression in the AG group, the samples in the GHNF group were designated as a calibrator. 18S was used as a housekeeping gene.
(AG; n = 5, GHNF; n = 5). The relative gene expression was determined using a TaqMan array 96-well FAST plate (4413257, Applied Biosystems; Bedford, Massachusetts, US). A TaqMan array plate contains 46 target genes and two assays for candidate endogenous control genes (
5
Quantifying Transcripts from 3T3-L1 Cells
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We isolated messenger RNA (mRNA) from 3T3‐L1 cells, cultured it with L‐14 extract using the PureLink™ RNA minikit (Invitrogen) and reverse‐transcribed it into complementary DNA (cDNA) using a cDNA kit (Promega). Next, the cDNA was amplified using a TB green mix (TAKARA) using the StepOnePlus™ Real‐Time polymerase chain reaction (PCR) System (Applied Biosystems) and analysed. The primers used for quantitative reverse transcription PCR (qRT‐PCR) were designed as the listed sequences (Table S1 ).
6
Quantitative Analysis of miR-33a/b
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Total RNA was isolated using TRIzol reagent (Invitrogen). MiR-33a/b were measured in accordance with the TaqMan MicroRNA Assays (Applied Biosystems) protocol, and the products were analyzed using a thermal cycler (Applied Biosystems StepOnePlus real-time polymerase chain reaction [PCR] system). Samples were normalized by U6 small nuclear RNA expression.
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