Total cytoplasmic RNA was isolated from the cells/tissues using TRIzol reagent (Invitrogen). The cDNA was synthesized from RNA templates using Moloney murine leukemia virus-derived reverse transcriptase (Promega, Madison, WI, USA). Primers for detecting proximal or distal Runx1/Runx3 transcripts were purchased from Eurofins Genomics (Tokyo, Japan), whereas primers for keratin 1, keratin 10, and hypoxanthine phosphoribosyltransferase (HPRT) transcripts were from Applied Biosystems (Foster City, CA, USA). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Invitrogen. To detect Runx1, Runx3, and GAPDH transcripts, semi-quantitative PCR was performed using a StepOne™ RT-PCR System (Applied Biosystems) and a Universal Probe Library Set (Roche Applied Science, Indianapolis, IN, USA). To detect keratin 1, keratin 10, and HPRT transcripts, real-time PCR was performed using TaqMan Fast Advanced Master Mix on a StepOne RT-PCR System (Applied Biosystems). PCR conditions were as follows: for Runx1, 39 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 40 s; for Runx3, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s; for GAPDH, 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s; for keratin 1, keratin 10, and HPRT, 40 cycles of 95 °C for 1 s, 60 °C for 20 s. The cDNA quantity in each sample was normalized relative to HPRT as a control.
Stepone rt pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, China
The StepOne RT-PCR system is a real-time PCR instrument designed for gene expression analysis, genotyping, and other applications. It features a compact, easy-to-use design and provides accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Trizol reagent, by Takara Bio (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Stepone software, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taq reagent kit, by Takara Bio (2 mentions)
Iscript supermix, by Bio-Rad (2 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (2 mentions)
Prism 7, by GraphPad (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Primescript rt pcr kit, by Takara Bio (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Rt2 first strand kit, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
67 protocols using stepone rt pcr system
1
Quantitative Analysis of Runx Transcripts
2
Quantitative PCR for Amyloid-Beta Protein
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For PCR using Taqman system, Taqman Universal PCR Master Mix was used with customized gene assays AJX00TP (Abeta) and Ce02507510_s1 (act-2: β-actin; Applied Biosystems, Foster City, USA) were used with the (Applied Biosystems, Foster City, USA). Cycling conditions were 50 °C × 2 min, then 95 °C × 10 min, followed by 40 cycles of 95 °C × 15 sec +60 °C × 1 min.
For PCR using SyBr system, QuantiFast SyBr Green PCR kit (Qiagen, Venlo, Netherlands) was used with self-design Abeta primers (FP: 5′ GAT GCA GAA TTT CGA CAT GAT TCA GG; RP: 5′ TCA AGC AAT GAC AAC TCC TCC C). Cycling conditions were 95 °C × 7 min, followed by 40 cycles of 95 °C × 10 sec + 60 °C × 1 min. All reactions were performed using a StepOne RT-PCR System (Life Technologies, Carlsbad, USA).
For PCR using SyBr system, QuantiFast SyBr Green PCR kit (Qiagen, Venlo, Netherlands) was used with self-design Abeta primers (FP: 5′ GAT GCA GAA TTT CGA CAT GAT TCA GG; RP: 5′ TCA AGC AAT GAC AAC TCC TCC C). Cycling conditions were 95 °C × 7 min, followed by 40 cycles of 95 °C × 10 sec + 60 °C × 1 min. All reactions were performed using a StepOne RT-PCR System (Life Technologies, Carlsbad, USA).
3
Real-Time qPCR Gene Expression Analysis
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Treated and untreated cells or tumor tissues were respectively harvested. The total RNA was extracted by the TRIzol reagent (Roche, Suisse). Then, the samples were treated with DNase I and reverse transcribed using oligo-dT primers. The total cDNA was used as starting material for real-time PCR (RT-PCR) with FastStart Universal SYBR Green Master Mix (Roche Applied Science, Germany) on a StepOne RT-PCR System (Life Technologies Corp., CA, USA). The reaction conditions were: 35 cycles at 95 °C for 30 s; 64 °C for 25 s; and 72 °C for 30 s. The specific primers reported in Table 1 were designed using gene runner software and synthesized by Beijing Aoke Biotechnology Co., Ltd. (Beijing, China). GAPDH expression was used to normalize the Cq values. Cq value was obtained from instrument software. The relative quantification of gene expression was calculated by 2-ΔΔCq method.
4
Quantitative Gene Expression Analysis
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Total RNA was extracted from the tumor tissues and cells using TRIzol reagent (Takara, Japan), and reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, Japan). Real-time PCR (RT-PCR) was performed at a final reaction volume of 20 μL with 1 μL of template cDNA at a concentration of 20 ng/μL, using the SYBR Premix Ex Taq Reagent Kit (Takara, Japan) on the StepOne RT-PCR System (Life Technologies, USA), according to the manufacturer’s instructions. The mRNA levels were normalized to β-actin levels. The primer sequences used for RT-PCR in the present study are listed in Supplementary Table 2
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from H9c2 cells and NRVMs, using TRIzol reagent. Two micrograms of RNA was reverse-transcribed with SuperScript II using random primers. Real-time PCR was performed using SYBR Green PCR Master Mix (Life Technologies) in an Applied Bio system's Step one RT-PCR system with the following thermal cycling conditions: 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min for denaturation, annealing and elongation. Relative mRNA levels were calculated by the method of 2−DDCt (Livak and Schmittgen 2001 (link)). Data were normalized to GAPDH mRNA levels. To determine the specificity of amplification, melting curve analysis was applied to all final PCR products. All samples were performed in triplicate. The expression suite software (Applied Bio-systems) was used to analyze the data.
6
RNA-Seq Library Preparation and Sequencing
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Total RNA was isolated using TRIzol reagent (Invitrogen). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies), and the RNA was quantified using an ND-2000 Spectrophotometer (Thermo). Libraries were prepared from the total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (New England BioLabs). mRNA was isolated using the Poly(A) RNA Selection Kit (Lexogen) and was used for cDNA synthesis and shearing, following the manufacturer’s instructions. Indexing was performed using Illumina indices 1–12. The libraries were enriched using PCR. Fragment size was evaluated using the Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit). Quantification was performed using a library quantification kit and a StepOne RT‒PCR System (Life Technologies). High-throughput sequencing was performed as paired-end 100 sequencing using a NovaSeq 6000 instrument (Illumina).
7
TBEV RNA Detection by RT-PCR
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Detection of TBEV RNA was assessed as previously described [57 (link)] with these minor changes: RT-PCR was performed with TaqMan Fast Virus 1-step mastermix (Applied Biosystem, Life Technologies) using the StepOne RT-PCR system (Life Technologies) according to the manufacturer’s instruction. To ensure adequate RNA extraction from the samples, human B-actin (Applied Biosystems, Life Technologies) was assayed in parallel as an endogenous control.
8
Tumor RNA Extraction and Real-Time PCR
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The RNA was extracted from the tumor samples and cells using TRIzol reagent (Takara, Japan), and cDNA was synthesized with a PrimeScript RT Reagent Kit (Takara, Japan). A real-time PCR (RT-PCR) was performed using the SYBR Premix Ex Taq Reagent Kit (Takara, Japan) with the StepOne RT-PCR System (Life Technologies, USA) according to the manufacturer's instructions. The mRNA levels were normalized to β-actin levels. The primer sequences used in the present study are listed in Supplementary Table 1.
9
Sural Nerve RNA Extraction and qPCR Analysis
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Sural nerve RNA was isolated using a RNeasy fibrous tissue mini kit (74704; Qiagen). cDNA template was generated using 200 ng of total RNA and iScript Supermix (1708840; BioRad) in a 40 μl reaction. The reaction was diluted 1:1 with dH2O. qPCR reactions were performed in triplicate using TaqMan™ 2X gene expression Master Mix (4369016; ThermoFisher/Applied Biosystems), 2 μl of template, and sequence-specific TaqMan™ probes (ThermoFisher/Applied Biosystems; Supplemental Table S7) in an Applied Biosystems StepOneTM RT-PCR system. The conditions used were: 40x cycles of 30 s at 95º C, 60 s at 55–60º C, and 30 s at 72º C. This was followed by a 5 min final phase at 72º C. CT values were used to calculate ΔCT and ΔΔCT (with YHWAZ as a housekeeping control and regenerators as the relative control group; Supplemental Table S7). Data are expressed as the mean of the relative quantity of gene expression (2−ΔΔCT) [88 ]. Statistically significant differences were calculated using a t-test with GraphPad Prism 7 software (GraphPad software Inc.).
10
Validation of Differential Gene Expression
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Two shared genes between DMGs and DEGs (Thy1 and PTGS1) were selected for validation by quantitative real-time polymerase chain reaction (RT-qPCR). RNA was isolated from sural nerve biopsies using RNeasy fibrous tissue mini kit (Qiagen, Valencia, CA, USA) from group 1 (n = 9–10) and group 2 (n = 10), which was used as the relative control group (set to 100%). Reverse transcription was performed using iScript Supermix (Bio-Rad Laboratories, Hercules, California). qPCR reactions were carried out using sequence-specific TaqMan™ probes (ThermoFisher/Applied Biosystems) for Thy1 (Hs06633377_s1) and PTGS1 (Hs00377726_m1) in an Applied Biosystems StepOneTM RT-PCR system. Using the 2−ΔΔCT method, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ) was used as the endogenous control and group 2 as the relative control. Statistically significant differences were calculated using a Student’s t test with the GraphPad Prism 7 software (GraphPad Software Inc.).
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