Total RNA from the fresh PSCC samples and paired adjacent tissues was extracted with the E.Z.N.A.® Total RNA kit II (cat. no., R6934-02; Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer's protocol. RT was performed using the RevertAid First Strand cDNA synthesis kit (cat. no., K1622; Thermo Fisher Scientific, Inc.; Waltham, MA, USA). qPCR was performed using SuperRealPreMix Plus with SYBR Green (cat. no., FP205-02; Tiangen Biotech Co., Ltd., Beijing, China) on the ViiA7 DX Real-Time PCR system (Thermo Fisher Scientific, Inc.) as follows: 95°C for 15 min followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 32 sec. The sequences of primers for CD109 and GAPDH are included in Table I . All PCR reactions were repeated in triplicate. CD109 gene expression was assessed by comparing the relative expression level of CD109 with the internal reference GAPDH by the 2−ΔΔCq method (20 (link)).
Viia 7 dx real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in China, Japan, United States
The ViiA 7 Dx Real-Time PCR System is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It provides accurate and reliable detection and quantification of nucleic acid targets.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Tiangen Biotech (1 mentions)
E z n a total rna kit 2, by Omega Bio-Tek (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Reverse transcription system, by Promega (1 mentions)
Wizard genomic dna purification, by Promega (1 mentions)
Primescript rt regent kit, by Takara Bio (1 mentions)
Sybr green kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
9 protocols using viia 7 dx real time pcr system
1
CD109 Expression in PSCC Samples
2
Quantitative Analysis of Gene Expression
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Total RNA from cells or fresh lung adenocarcinoma tissue was extracted with the E.Z.N.A.TM Total RNA Kit ii (OMEGA Bio-tek, Winooski, VT, USA) according to the manufacturer's instructions. Reverse transcription was performed with the Revert Aid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). QRT-PCR was carried out using SYBR Green (Tiangen Biotech, Beijing, China) using the ViiA 7 DX Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were as follows: 50°C 2 min; 95°C 2 min; 95°C 15 s, 40 cycles; 60°C 1 min. GAPDH levels were used for normalization during the quantitative measurement of gene expression. All PCR reactions were repeated in triplicate. The sequences of primers used are summarized in Table 2 .
3
Quantifying Endothelial Gene Expression
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Confluent HUVECs on 6-well plates were treated with 1 mmol catalpol and collected at 24 h. Total RNA was extracted with the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.). qPCR was conducted using SYBR Premix Ex TaqII (Takara Bio, Inc., Shita, Japan) on the ViiA 7 DX Real-Time PCR System (Thermo Fisher Scientific, Inc.). The sequences of primers for ERG, CDH5 (encoding VE-cadherin) and OLDN (encoding occludin) genes are shown in Table I. The reaction conditions were as follows: 94˚C for 5 min, 30 cycles of 94˚C for 30 sec, 57˚C for 30 sec and 72˚C for 2 min, then a final extension at 72˚C for 5 min. All PCR reactions were repeated in triplicate. Gene expression was assessed by comparing the relative expression level of each gene with the internal reference GAPDH using the ΔΔ Ct method.
Statistical analysis. Statistical significance was assessed using paired-sample t-tests. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc, Chicago, IL, USA). All experiments were repeated three times unless otherwise stated.
Statistical analysis. Statistical significance was assessed using paired-sample t-tests. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc, Chicago, IL, USA). All experiments were repeated three times unless otherwise stated.
4
Quantitative RNA Analysis Using SYBR Green
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Total RNA was extracted from tissues and cells with TRIzol reagent (Takara, Japan) according to manufacturer's instruction. Reverse transcription was conducted using the PrimeScript RT Reagent Kit (Takara). Real-time quantitative PCR was performed on triplicate samples in a reaction mix of SYBR Green (Takara) with ViiA 7 Dx Real-Time PCR System (Applied Biosystems). The mRNA levels were normalized against β-actin. Sequences of primers used for qRT-PCR in this study were listed in Supplementary Table 3 .
5
Quantitative RT-PCR for Gene Expression
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Total RNAs were extracted by miRNeasy Mini Kit (Qiagen) and reverse transcribed by SuperScript® III First-Strand Synthesis System (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed with the ViiA 7DX real-time PCR system (Applied Biosystems) using the Fast SYBRGreen Master Mix (Applied Biosystems). Each reaction was performed in triplicate using 0.5 μL of cDNA in a final volume of 8 μL. Relative transcript levels of target genes were normalized with GAPDH mRNA levels. The qPCR primers used in this study are listed in Table1 .
6
Quantitative RT-PCR Analysis of Nogo-B and TGFβ1
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Total cellular RNA was isolated from cells using the RNeasy Micro kit (Qiagen, Germantown, MD, USA). First strand cDNA synthesis was performed using the Reverse Transcription System (Promega, Madison, WI, USA). Quantitative real-time PCR was performed using a SYBR Green PCR Kit (Applied Biosystems, Foster City, CA, USA) and the ViiA 7 Dx Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The sequences of the primers used in this study are: Nogo-B-F:5′-GCAGTGTTGATGTGGGTATTT-3′; Nogo-B-R:5′-CTGTGCCTGATGCCGTTC-3′; TGFβ1-F:5′-GTACCTGAACCCGTGTTGCT-3′; TGFβ1-R:5′-TGAACCCGTTGATGTCCACT-3′; β-actin-F:5′-AATCGTGCGTGACATTAAGGAG-3′; β-actin-R: 5′-ACTGTGTTGGCGTACAGGTCTT-3′. Each reaction was performed in triplicate.
7
Cord Blood mtDNA Quantification Protocol
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Cord blood was collected immediately at delivery. Blood samples were centrifuged and placed at -80 °C until DNA extraction. DNA was isolated from the leukocytes of umbilical cord blood samples by Wizard ® Genomic DNA Purification (Promega Corporation, Madison, WI, USA). Relative cord blood mtDNAcn was measured by quantitative real-time PCR (qPCR) assay and the sequences of primer, reaction mixture, and PCR thermal cycling profile are described previously (Liu et al., 2019; (link)Wu et al., 2019) (link). In brief, relative cord blood mtDNAcn was calculated by the ratio of the mitochondrial gene copy numbers (mtND1) to the single-copy nuclear control gene [human beta-globin (hbg)]. All measurements were conducted in triplicates using the ViiA 7 Dx Real-Time PCR System (Applied Biosystems) in 384-well plates. A pool of 50 genomic DNA samples selected randomly from our study population was used to construct a standard curve with five-point serial dilution, ranging from 104 ng/L to 0.4 ng/L (R 2 ≥ 0.99).To ensure quality control, each plate included standard curve, negative controls, and inter-plate controls. The intra-run and inter-run coefficients of variation for mtDNAcn measurements were 2.8% and 3.8%, respectively.
8
RNA Extraction and qPCR Analysis in BAT
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BAT samples were minced using a homogenizer (IKA, Staufan, Germany), and total RNA was isolated from these samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of the extracted RNA were assessed with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, complementary DNA was acquired from reverse transcription of 500 ng RNA using the PrimeScript™ RT regent kit (Takara, Mountain View, CA, USA). All primers were designed online with Primer 3 (http://sourceforge.net/projects/primer3/ ), and the uniqueness of their amplification products was checked using the Basic Local Alignment Search Tool of NCBI. The sequences of these primers are listed in Table S2 . The qPCR analyses were performed on the ViiA™ 7 DX Real-Time PCR System (Applied Biosystem) with a SYBR Green Kit (TaKaRa, Tokyo, Japan), and β-actin was used as the internal control.
9
Quantitative Analysis of DDAH1 and DDAH2
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Total mRNA from cell samples was extracted using Trizol reagent (Takara, Japan) as described elsewhere according to the manufacturer's protocol. cDNA was generated from total RNA using reverse transcription kits (Takara, Japan). Amplification was performed by using ViiA 7 DX real-time PCR system (Applied Biosystem, USA) with SYBR real-time PCR kit (Takara, Japan) and the amplification condition was performed with an initial step at 95°C for 30 s and 40 cycles at 95°C for 5 s, annealing at 60°C for 31 s for each target gene. Results were expressed as the ratios of target genes against glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA for DDAH1 and DDAH2 and the small nuclear RNA U6 for miR-21. The primers for DDAH1, DDHA2, and GAPDH were from Shanghai Sangon Company (Shanghai, China), while the primers for miR-21 and U6 were purchased from RiboBio (Guangzhou, China). The primers used in the amplification were as follows: DDAH1-F, 5′-GCCTGATGACATAGCAGCAA-3′, DDAH-1-R, 5′-CCATCCACCTTTTCCAGTTC-3′; DDAH2-F: 5′-ACAAGGACCCCCGCTAAAA-3′, DDAH2-R, 5′-AAGGGAGTCCCCGTCTTCAA-3′; GAPDH-F, 5′-CTGCACCACCAACTGCTTAG-3′, GAPDH-R, 5′-AGGTCC-ACCACTGACACGTT-3′.
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