Quantitative real-time QPCR (RT-QPCR) was performed in duplicates on RNA specimens prepared in independent experiments. All transcript levels were normalized to GAPDH expression of each sample. Primer sequences were as follows: p62, 5’-CACCTGTCTGAGGGCTTCTC-3’ (sense) and 5’- CACACTCTCCCCAACGTTCT-3’ (antisense); GAPDH, 5’-GGTATCGTGGAAGGACTCATGAC-3’ (sense), 5’-ATGCCAGTGAGCTTCCCGT-3’ (antisense). The PCR conditions were as follows: initial denaturation for 10 min at 95°C, followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed on an ABI PRISM 7900 HT instrument (Applied Biosystems, Foster City, CA, USA). A mean cycle of threshold (Ct) value for each duplicate measurement was calculated.
Abi prism 7900ht instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The ABI PRISM 7900HT instrument is a real-time PCR system designed for high-throughput gene expression analysis. It features a high-performance optical system, advanced thermal cycling capabilities, and intuitive software for data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Sds v2, by Thermo Fisher Scientific (4 mentions)
Taqman 5 exonuclease assay, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (3 mentions)
Sds 2, by Thermo Fisher Scientific (2 mentions)
384 well pcr plate, by Thermo Fisher Scientific (2 mentions)
High capacity reverse transcription cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Uracil n glycosylase, by Thermo Fisher Scientific (2 mentions)
Taqman universal pcr master mix 2, by Thermo Fisher Scientific (2 mentions)
Sequencher v5, by Gene Codes (1 mentions)
Puregene kit, by Qiagen (1 mentions)
Abi prism 3130xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
37 protocols using abi prism 7900ht instrument
1
Quantitative Real-Time PCR Analysis
2
Quantitative RT-PCR for IL-6 Expression
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Each gene expression was quantified by real-time PCR using a SensiMix SYBR Kit (Bioline Ltd., London, UK) on an ABI PRISM 7900HT instrument (Applied Biosystems, CA, USA). Sequences of the primer sets used in this study were as follows. IL-6: forward, 5′-AAT TCG GTA CAT CCT CGA CGG-3′ and reverse, 5′-GGT TGT TTT CTG CCA GTG CC-3′; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, used as an endogenous control): forward, 5′-ATT GTT GCC ATC AAT GAC CC-3′ and reverse, 5′-AGT AGA GGC AGG GAT GT-3′. All reactions were performed in triplicate. Data were analyzed and normalized to the expression of the housekeeping gene GAPDH as an internal control. Fold change of the target gene was evaluated by the comparative CT method (2−∆∆CT).
3
Quantitative Analysis of miRNA Expression
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Total RNA was extracted from cell lines using the TOOLSmart RNA Extractor (Biotools, Taipei City, Taiwan) according to the manufacturer’s instructions. Approximately 350 ng of the total RNA from each sample and the expression levels of miR-143-3p in both cell lines and FFPE tissues were analyzed using the TaqMan MicroRNA Assay (assay ID: 002249). The expression levels of hsa-miR-16 (TaqMan MicroRNA Assay ID: 000008) were used as an internal control. RNA samples were subjected to real-time qPCR. Transcription levels were normalized to the GAPDH values of each sample. The primer sequences were as follows: BIRC2, 5′-GGAGATGATCCATGGGTAGA-3′ (sense), 5′-ACAAACTCTTGGCCTTTCAT-3′(antisense); GAPDH: 5′-GGTATCGTGGAAGGACTCATGAC-3′ (sense), 5′-ATGCCAGTGAGCTTCCCGT-3′ (antisense); and 18S rRNA, 5′-AAACGGCTACCACATCCAAG-3′ (sense), 5′-CCTCCAATGGATCCTCGTTA-3′ (antisense). Amplification was performed as follows: initial denaturation (10 min) at 95 °C, followed by 45 cycles at 95 °C for 15 s and 45 cycles at 60 °C for 1 min using an ABI PRISM 7900 HT instrument (Applied Biosystems, Foster City, CA, USA). All measurements were performed in duplicate, and the threshold cycle Ct was determined.
4
Genetic Variant Screening in Leukocyte DNA
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Genomic DNA from control samples was extracted from peripheral leukocytes using the Puregene Kit according to the manufacturer’s instructions (Gentra Systems, Inc., Minneapolis, MN, USA). Mutation screening was achieved through Sanger sequencing. Reference sequence NM_020193.3 was obtained from the USCS genome browser [32 , 33 (link)]. The genome build GRCh37 (hg19) was used. Whole coding regions and exon-intron boundaries were analysed. EMSY primer sequences and PCR conditions are available upon request. Sequencing was performed using the Big Dye Terminator v.3.1 Cycle Sequencing Kit and the ABIPRISM 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). The sequences were analysed using Sequencher v.5.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). The RefSNP number for the identified variants was obtained from the NCBI Single Nucleotide Polymorphism database (dbSNP) [34 ]. Two variants, rs1044265 and rs2513513, were analysed from controls using TaqMan SNP Genotyping Assays according to the manufacturer’s instructions with the ABI Prism® 7900HT instrument and SDS2.2.2 software (Applied Biosystems). The genotyping call rates of the SNPs were ≥0.98.
5
Quantitative Real-Time PCR Analysis
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Through the use of TRIZOL™ (Catalog number: 15596026; Invitrogen), total RNA from target tissues or cells was isolated. cDNA was synthesized using an oligo ethylene glycol‐based transcript first‐strand cDNA synthesis kit (Product No.: 04896866001; Roche Diagnostics, Basel, Switzerland). For cDNA synthesis, 500 ng of total RNA was used, and the total RNA was diluted with RNAse‐free H2O, and the total RNA's final concentration was adjusted to 5 ng/mol. RT‐qPCR detection was performed using a Power SYBR Green PCR master mix (Catalog number: 4367659; Life Technologies) in an ABI Prism 7900HT instrument (Applied Biosystems). The target gene's relative expression level was calculated using the 2−ΔΔCt method. The β‐actin level level (for mRNA expression) was used as an internal reference. The primers were listed in Table 1 .
6
SNP Genotyping of ZNF804A Variants
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Genomic DNA was extracted from buccal mucosa on a cotton swab using the Real Extraction DNA kit (Durviz S.L.U., Valencia, Spain). The two intronic single nucleotide polymorphisms of the ZNF804A included in the study (rs7597593 and rs1344706; D’ = 0.94 r2 = 0.32) were genotyped using TaqMan 5’ exonuclease assay (Applied Biosystems).
The final volume was 5 μL, which contained 5 ng of genomic DNA, 2.5 μL of TaqMan Master Mix, and 0.125 μL of 40x genotyping assay (assays C_223561_10 and C_2834835_10, respectively). The cycling parameters were as follows: 95°C for 10 min followed by 40 cycles of denaturation at 92°C for 15s and annealing/extension at 60°C for 1 min. Polymerase chain reaction plates were read on an ABI PRISM 7900HT instrument and SDS v2.1 software (Applied Biosystems) was used for the genotype analysis of data. Both polymorphisms were in Hardy-Weinberg equilibrium. For accuracy of genotyping, 20% of the samples (chosen randomly) were genotyped twice, showing concordant genotypes.
The final volume was 5 μL, which contained 5 ng of genomic DNA, 2.5 μL of TaqMan Master Mix, and 0.125 μL of 40x genotyping assay (assays C_223561_10 and C_2834835_10, respectively). The cycling parameters were as follows: 95°C for 10 min followed by 40 cycles of denaturation at 92°C for 15s and annealing/extension at 60°C for 1 min. Polymerase chain reaction plates were read on an ABI PRISM 7900HT instrument and SDS v2.1 software (Applied Biosystems) was used for the genotype analysis of data. Both polymorphisms were in Hardy-Weinberg equilibrium. For accuracy of genotyping, 20% of the samples (chosen randomly) were genotyped twice, showing concordant genotypes.
7
Quantitative RT-PCR for LTβ and VEGF-C Expression
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Total RNA was isolated from salivary glands with an RNeasy mini kit (Qiagen) and the RNA was then reverse transcribed using a high-capacity reverse transcription cDNA synthesis kit (Applied Biosystems) according to the manufacturer’s specifications. Reverse transcription was carried out on Techne 312 thermal cycler PCR machine. Quantitative RT-PCR (Applied Biosystems) was performed on cDNA samples for LTβ and VEGF-C mRNA expression. β-Actin was used as an endogenous control. The primers and probes used were from Applied Biosystems. The quantitative real-time PCR was run in duplicates on a 384-well PCR plate (Applied Biosystems) and detected using an ABI Prism 7900HT instrument. Results were analyzed with the Applied Biosystem’s SDS software (SDS 2.3). We used the mean of two technical replicates (Ct values) to calculate the ΔCt value. The Ct of the β-actin was subtracted from the target gene Ct value and the relative amount was calculated as 2−ΔCt. The relative quantity (RQ) expression values were calculated as 2−ΔΔCt, where ΔΔCt is the difference between the ΔCt values of cannulated salivary glands and the ΔCt of noncannulated salivary glands. Ct values >34 were not accepted, nor were technical replicates with more than two cycle differences between them.
8
Quantitative Real-Time RT-PCR Analysis
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Total RNA was isolated using ISOGEN II reagent (Nippon Gene), according to the manufacturer’s protocol. cDNA was created from 0.5 μg total RNA using an oligo(dT) primer (Life Technologies), random hexamers (Takara), and M-MuLV reverse transcriptase (NEB). Real-time RT-PCR using reagents containing SYBR green was performed with an ABI PRISM 7900HT instrument (Applied Biosystems). Expression levels were compared with known standard samples and normalized to GAPDH. Primer sequences are shown in Table S2 .
9
Quantitative Real-Time PCR Analysis of Salivary Gland Transcripts
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Quantitative real-time PCR was performed as previously described in8 (link),52 (link) using following primers Ccl19 (Mm00839967_g1), Cxcl13 (Mm00444533_m1), Lta (Mm00440229_g1), Ltb (Mm00434774_g1), Tnf (Mm00443258_m1), Tnfrsf1a alias Tnfra (Mm00441883_g1), Icos (Mm00497600_m1), Icosl (Mm00497237_m1) and Actb alias β-actin (Mm01205647_g1) was used as an endogenous control. Briefly, total RNA was isolated from salivary glands with an RNeasy mini kit (Qiagen) and the RNA was then reverse-transcribed using the high capacity reverse transcription cDNA synthesis kit (Applied Biosystems) according to the manufacturer’s specifications. Reverse transcription was carried out on Techne 312 Thermal Cycler PCR machine. Quantitative RT-PCR (Applied Biosystems) was performed on cDNA samples with the indicated primers and was run in duplicates on a 384-well PCR plate (Applied Biosystems) and detected using an ABI PRISM 7900HT instrument. Results were analyzed with the Applied Biosystems SDS software (SDS 2.3).
10
RT-qPCR Analysis of Gene Expression in Breast Cancer Cells
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Total RNA extracted from human breast cancer cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) were used for RT-qPCR analysis. In brief, 1 µg total RNA from each sample was reverse transcribed (denaturation, 94°C for 30 sec; annealing 58°C for 30 sec and extension 72°C for 45 sec) using a RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Inc.). Alteration of gene expression was performed using SensiMix SYBR kits (Bioline) and ABI PRISM 7900HT instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primer sequences were as follows: human PDGFB (forward, 5′-CGAATGGTCACCCGAGTTTG-3′ and reverse, 5′-GAGATGCTGAGTGACCACTC-3′), human ER-α (forward, 5′-CGCTACTGTGCAGTGTGCAAT-3′ and reverse, 5′-CCTCACAGGACCAGACTCCATAA-3′) and GAPDH as an endogenous control (forward, 5′-ATTGTTGCCATCAATGACCC-3′ and reverse, 5′-AGTAGAGGCAGGGATGT-3′). Thermocycling conditions were 95°C for 10 min, 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 15 sec. For data analysis, the raw threshold cycle (CT) value was normalized to the housekeeping gene for each sample to obtain ΔCT. Normalized ΔCT was calibrated to control cell samples to calculate ΔΔCT (13 (link),14 (link)).
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