Total RNA was isolated from blood and tissues using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instructions. cDNA for miRNA analysis was synthesized from 5 ng total RNA using the Universal cDNA synthesis kit II (Exiqon). For the determination of miRNA expression, qPCR was performed using the miRCURY LNA Universal RT micro RNA PCR LNAPCR primer sets for miR-16-5p, miR-130a-3p, miR-17-5p, miR-103-3p, miR-30c-5p, and miR-21a-5p (Exiqon), and the SYBR green PCR kit (Exiqon) in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific). cDNA for mRNA analysis was synthesized from 1 μg total RNA using PrimeScript 1st strand cDNA Synthesis Kit (Exiqon). For the determination of mRNA expression, qPCR was performed using gene-specific primer pairs and the Roche SYBR-Green master mix in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific).
Abi 7900ht real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Japan, Switzerland
The ABI 7900HT Real-Time PCR system is a high-throughput, real-time PCR instrument designed for accurate and reliable gene expression analysis. It provides precise quantification of DNA and RNA targets in a multi-well format.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (110 mentions)
Primescript rt reagent kit, by Takara Bio (44 mentions)
Trizol, by Thermo Fisher Scientific (21 mentions)
Sybr premix ex taq kit, by Takara Bio (18 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Sds 2, by Thermo Fisher Scientific (12 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (11 mentions)
Trizol regent, by Thermo Fisher Scientific (10 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (9 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Sybr green master mix, by Thermo Fisher Scientific (8 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (8 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (6 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Primescript rt master mix, by Takara Bio (6 mentions)
Taqman assay, by Thermo Fisher Scientific (6 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
334 protocols using abi 7900ht real time pcr system
1
Quantification of miRNA and mRNA Expression from Blood and Tissues
2
Quantifying miR-150 Expression in ADSCs
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Total RNA was extracted from ADSCs using Trizol (Catalogue Number: 15596018, Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized using the RiboTM Reverse Transcription Kit (Catalogue Number: C10170, RiboBio, China). The reverse transcription reaction for miR-150 was carried out with Bulge-LoopTM miRNA qRT-PCR Primers (RiboBio, China). Relative expression was determined using U6 (RiboBio, China) as an internal control for miR-150. Quantitative real-time PCR was performed with three technical replicates on the ABI-7900HT Real-Time PCR System (Applied Biosystems) using the SYBR Green Master Mix (Catalogue Number: RR036A, Takara, Dalian, China).
3
Pharmacogenetic Analysis of SNP Markers
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The SNPs included in this study were selected based on previous studies that demonstrated their association with pharmacosensitivity (GSTP1 rs1695, GSTO1 rs4925, GSTO2 rs156697, ABCB1 rs3747802, and ABCB1 rs3213619). Genomic DNA of each individual was extracted from 150 μL of EDTA-anticoagulated peripheral blood samples by using a DNA extraction kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions. The polymorphisms were genotyped using a TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA) and a 384-well ABI 7900HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). A 1-μg sample of total DNA was used for genotyping with primers (Invitrogen, Karlsrule, Germany) (shown in the Supplementary data ). SDS 2.4 software (Applied Biosystems, Foster City, CA, USA) was used for allelic discrimination. Each sample was run in triplicate. For quality control, four negative controls were included in each plate, and 5% of the samples were randomly selected for repeated genotyping to verify the results; all of the results were 100% consistent. Primers, probes, and reaction conditions for each SNP analysis are available upon request. Amplification was performed under the following conditions: 50 °C for 2 min; 95 °C for 10 min; and 45 cycles of 95 °C for 15 s and 60 °C for 1 min.
4
Melanoma Tissue RNA Extraction and qRT-PCR Analysis
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Melanoma tissues samples were extracted from patients diagnosed with melanoma by (three independent) experienced physicians (based on Chinese guidelines for diagnosis and treatment of melanoma). The total RNA was extracted using the Trizol Reagent (Invitrogen) from tissues based on the manufacturer’s instructions (Trizol, chloroform, and isopropanol were added in turn; the supernatant was centrifuged and quantified by absorbance value of 260nm and stored at - 80 °C). Subsequently, a reverse transcription kit (Takara Bio, Inc., Otsu, Japan) was used to reverse-transcribe RNA into cDNA in a 20ul system. Subsequently, the cDNA was used as a template, detected by the SYBR Green (Takara Bio) and ABI 7900HT Real‑Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers used are shown in Table S1
5
Liver Gene Expression Analysis
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We analyzed by qRT-PCR the levels of expression of the following genes: LDLR, HMG-CoAR, ACAT2, CYP7A1 and TNFα. All the used primer sequences were listed in S1 Table . The total RNA was extracted from the mouse liver tissue using TriZol (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions, and the genomic DNA was removed by treating with DNAse enzyme. The RNA concentration and the purity were measured using NanoDrop 2000 (Thermo Scientific). RNA (1 μg) was converted to cDNA by reverse transcription using iScript cDNA synthesis kit (Bio-Rad).
qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad) and the ABI 7900 HT Real Time PCR System (Applied Biosystems). The qRT-PCR results were normalized on expression levels of β-2–microglobulin, used as an internal reference. The results were expressed as relative gene expression levels by using the 2^-ΔCT formula.
qRT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad) and the ABI 7900 HT Real Time PCR System (Applied Biosystems). The qRT-PCR results were normalized on expression levels of β-2–microglobulin, used as an internal reference. The results were expressed as relative gene expression levels by using the 2^-ΔCT formula.
6
Quantifying miRNA Expression by RT-qPCR
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Total RNA was extracted from samples and cells by applying TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit was purchased from Beijing Transgen Biotech Co., Ltd. (Beijing, China). Then total RNA was converted into cDNA through RT according to the manufacturer's protocol. Following RT, qPCR was performed using the TransScript® Tip Green qPCR SuperMix kit according to the manufacturer's protocol (Transgen Biotech Co., Ltd.) with an ABI 7900HT Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following forward and reverse primers were used for RT-qPCR: miR-20a, forward 5′-TAAAGTGCTTATAGTGCAG-3′ and reverse 5′-TGCGTGTCGTGGAGTC-3′ (LNA); U6, forward 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse 5′-ACGCTTCACGAATTTGCGTGTC-3′. The PCR reaction mixtures contained 2× TransScript® Tip Green qPCR SuperMix (10 µl), 4 µM primers (2 µl), 1 µl cDNA and 7 µl ddH2O in a total volume of 20 µl. The PCR thermocycling conditions were as follows: 94°C for 30 sec, and 40 cycles of 5 sec at 94°C and 30 sec at 60°C. The comparative 2−ΔΔCq method (26 (link)) was used for relative quantification and statistical analysis.
7
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and concentration were determined using the NanoDrop 2000 system (Thermo Fisher Scientific, Wilmington, DE, USA). The expression status and target genes and β-actin were determined by quantitative real-time polymerase chain reaction (PCR) using an ABI 7900HT Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using Power SYBR® Green PCR Master Mix (Invitrogen). The primers for KLF3 real-time PCR were 5′-TGTCTCAGTGTCATACCCATCT-3′ (forward) and 5′-CCTTCTGGGGTCTGAAAGAACTT-3′ (reverse). The primers for β-actin were 5′-CTACGTCGCCCTGGACTTCGAGC-3′ (forward) and 5′-GATGGAGCCGCCGATCCACACGG-3′ (reverse). All reactions were run in triplicate.
8
Quantifying MICAL2 Gene Expression in Colorectal Cancer
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Total RNA of 1×106 cells of MICAL2-knockdown HCT116 or MICAL2-knockdown SW480 was extracted using Trizol reagent (Life Technologies) following the manufacturer's instructions. 1 μg DNase-treated RNA was reverse transcribed using Revert AidTM First-Strand cDNA Synthesis Kit (MBI Fermentas, USA) according to the manufacturer's instructions. The threshold cycle (Ct) value of each sample was determined using Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) in ABI 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA). Sequences of primers used are shown in Table S3 . Relative mRNA expression of each target gene was normalized to the expression of the housekeeping gene GAPDH. Relative mRNA level was calculated as two power values of ΔCt (Ct value of GAPDH Ct of target gene).
9
Quantitative RT-PCR Analysis of TGM2 Expression
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Total RNA was extracted with RNAiso Plus (Takara, Tokyo, Japan). Reverse transcription was conducted using Takara PrimeScript™ RT Master Mix to obtain cDNA. Quantitative RT-PCR was performed with TB Green® Premix Ex Taq™ to determine the expression of candidate genes using an ABI 7900HT Real-Time PCR system (Applied Biosystems, CA, USA). The primer sequences are listed in Table 1 .
Primer sequences performed in the text
| TGM2 Forward | 5′-GAGGAGCGGCAGGAGTATG- 3′ |
| TGM2 Reverse | 5′-CAGGAACTTGGGGTTGACATC- 3′ |
| β-actin Forward | 5′-TTGTTACAGGAAGTCCCTTGCC- 3′ |
| β-actin Reverse | 5′-ATGCTATCACCTCCCCTGTGTG- 3’ |
10
Quantitative Analysis of Rat Pdrg1 and Mat1a
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RNA purification and analysis was carried out as previously described using 100–150 mg of rat tissues or H35 cells (4 x 105 cells)[6 (link), 32 (link)]. Gene specific primers for rat Pdrg1 were designed using the program Primer Express 3.0 (Applied Biosystems, Foster City, CA, USA) with Tm values between 58–60°C (sense 5’-GACCTGGACACCAAGAGGAA-3’ , antisense 5’-GGTGCTCCTGATCTTTCTGG-3’ ); Mat1a and 18s primers were previously described [32 (link)]. Reverse transcription and cDNA amplification were carried out as described [32 (link)], using 300 nM (Mat1a and Pdrg1) and 100 nM (18s) primer concentrations and Power SYBR Green PCR Master Mix (Applied Biosystems). Expression was evaluated using the ABI 7900HT Real-Time PCR system (Applied Biosystems) at the Genomic Service of our institute. Relative expression ratios were normalized to the geometric mean of the 18s gene used as a control. Experimental efficiencies were calculated for each transcript and used to obtain the fold changes according to Pfaffl et al. [33 (link)].
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