FBS, MTT, doxorubicin, anti‐actin antibody, and cell culture medium were purchased from Sigma Chemical Co. (St Louis, MO, USA). QuantiTect SYBR Green PCR Kit was obtained from Qiagen (Germantown, MD, USA). Antibodies against to E‐cadherin, CDH2, vimentin, ZEB1 and β‐catenin were obtained from Cell Signaling Technologies (Danvers, MA, USA). A Dual‐Luciferase Assay Kit, caspase‐3/7 activity assay kit and Lipofectamine 2000 were obtained from Promega (Madison, WI, USA). TRIzol and a BLOCK‐iT™ Pol II miR RNAi Expression Vector Kit were purchased from Invitrogen (Carlsbad, CA, USA). Opti‐MEM, a High‐Capacity cDNA Reverse Transcription Kit, a miRNA expression reporter vector, miR‐708‐3p mimics, antisense oligonucleotides of miR‐708‐3p (ASO miR‐708‐3p), negative control oligonucleotides (NC), and primer sets of RNU6 and miR‐708‐3p were purchased from Life Technologies (Carlsbad, CA, USA). Invasion Assay Kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA). In Situ Cell Death Detection kits were purchased from Roche (Penzberg, Germany).
Ahigh capacity cdna reverse transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The High-Capacity cDNA Reverse Transcription Kit is a laboratory tool used to convert RNA into complementary DNA (cDNA) molecules. This kit provides the necessary reagents and enzymes to perform the reverse transcription process, which is a fundamental step in various molecular biology applications such as gene expression analysis and cDNA library construction.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Mirneasy isolation kit, by Qiagen (2 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Block it pol 2 mir rnai expression vector kit, by Thermo Fisher Scientific (1 mentions)
Caspase 3 7 activity assay kit, by Promega (1 mentions)
Lipofectamine 2000, by Promega (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Viiatm 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Optical 384 well plates, by Thermo Fisher Scientific (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
15 protocols using ahigh capacity cdna reverse transcription kit
1
Molecular Mechanisms of miR-708-3p in Cancer
2
RNA Isolation and qRT-PCR Analysis Protocol
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Approximately 10 mg of muscle was homogenized, with one stainless steel bead (Tissue Lyser II, Qiagen, UK), for 2 min at frequency of 30 s−1 in 500 μL of TRizol (Life Technologies/Thermo Fisher Scientific) to isolate total RNA according to the manufacturer's instructions. A high‐capacity cDNA reverse transcription kit (Life Technologies) was used to reverse‐transcribe 500 ng of total RNA for quantitative reverse transcription PCR. Precisely 1 μL of 1:10 diluted cDNA was added in each well of 384‐optical well plates (Life Technologies). Exon–exon boundary‐specific primers were mixed with SYBR Select Master Mix (LifeTechnologies) and RNase‐free water, and 6 μL of the mixed solution, as well as 1 μL of each cDNA were added to each well, with samples run in triplicate. The ViiATM 7 Real‐Time PCR System (Life Technologies) was used according to the following thermal cycling conditions: 2 min at 50°C; 2 min at 95°C; 40 cycles of 15 s at 95°C; and 60 s at 60°C. The ΔΔCt method was used to quantify target mRNA expression with Peptidylprolylisomerase‐A levels measured to correct for variations in RNA input/cDNA synthesis. Primer sequences for each of the probed genes are listed in TableS1 .
3
Liver Gene Expression Analysis in Rats
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Frozen sections from the livers (on the 10th day of the experiment) were collected in lysis buffer. Total RNA was isolated with TRIzol (cat. no. 15596–018; Life Technologies). RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific); 1μg RNA per sample was converted into cDNA.
A high-capacity cDNA reverse transcription kit (4368814; Life Technologies) was used for cDNA synthesis as recommended by the supplier. PCR was performed by the QuantStudio™ 3 System (Thermo Fischer Scientific) sequence detection system, using Life Technologies TaqMan gene expression assays (TGR5: Rn01400316_s1; FXR: Rn00572658_m1; IL-6: Rn01410330_m1; HGF: Rn00566673_m1; SCF: Rn01502851_m1; CTGF: Rn01537279_g1; BIRC5: Rn00574012_m1; IFNG: Rn00594078_m1; TNFR1: Rn00565310_m1) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. All samples were run in triplicate, in a 20 μl reaction volume. Results were obtained as threshold cycle (CT) values. Expression levels were calculated using the ΔCT method. The values were calculated as the mean values of three independent measurements, and the expression levels of mRNA in all samples were defined as a ratio to GAPDH expression.
A high-capacity cDNA reverse transcription kit (4368814; Life Technologies) was used for cDNA synthesis as recommended by the supplier. PCR was performed by the QuantStudio™ 3 System (Thermo Fischer Scientific) sequence detection system, using Life Technologies TaqMan gene expression assays (TGR5: Rn01400316_s1; FXR: Rn00572658_m1; IL-6: Rn01410330_m1; HGF: Rn00566673_m1; SCF: Rn01502851_m1; CTGF: Rn01537279_g1; BIRC5: Rn00574012_m1; IFNG: Rn00594078_m1; TNFR1: Rn00565310_m1) according to the manufacturer’s instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. All samples were run in triplicate, in a 20 μl reaction volume. Results were obtained as threshold cycle (CT) values. Expression levels were calculated using the ΔCT method. The values were calculated as the mean values of three independent measurements, and the expression levels of mRNA in all samples were defined as a ratio to GAPDH expression.
4
Screening small-molecule NCI library
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The library of small‐molecule drugs (NCI Spectrum Compound Library 2400 chemical compounds dissolved at 1 mmol/L in DMSO) was purchased from the University of Rochester Pathway Discovery Resource. SAHA was purchased from L C Laboratories (#V‐8477, purity >99%; Woburn, MA). iQ SYBR Green Supermix (#1708886) was purchased from Bio‐Rad Laboratories (Hercules, CA). A High‐Capacity cDNA Reverse Transcription Kit (#4374966) was obtained from Thermo Fisher Scientific (Waltham, MA). Recombinant human tumor necrosis factor alpha (TNFα) was purchased from R&D Systems (#210‐TA; Minneapolis, MN). Lipofectamine 2000 Transfection Reagent was purchased from Thermo Fisher Scientific. The wild‐type and MEF2 binding site defective mutant KLF2‐luc promoter‐driven luciferase reporter (KLF2‐luc) plasmids were gifted by Prof Mukesh Jain.10 KLF2 adenovirus was from ViGene Biosciences, Inc (#VH849868; Rockville, MD).
5
Targeted gene expression analysis
6
cDNA Synthesis and qPCR Validation
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A High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to synthesize cDNA from 200 ng of total RNA. qPCR was performed on the cDNA using an ABI 7900 HT Fast Real-Time PCR System (Thermo Fisher Scientific). Raw threshold-cycle (Ct) values were obtained using SDS2.4 (Thermo Fisher Scientific). The relative quantification of gene transcripts was determined by the ddCt method. A standard curve was also generated to evaluate the efficiency of the qPCR experiment. Each sample was run in duplicate along with an endogenous control. We assessed the appropriate reference gene using TaqMan probes as endogenous controls. We selected GAPDH (Hs02758991_g1) as the reference gene for validation, including randomly selected genes. P-values were calculated by Student’s t-test. All primers used for the qRT-PCR analysis are listed in Supplementary Table S2 .
7
Liver RNA Expression Profiling in Mice
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Frozen sections from the livers of groups 1 and 2 mice were collected in lysis buffer. Total RNA was isolated with TRIzol (cat. no. 15596-018; Life Technologies). RNA concentration was measured by NanoDrop 1000 (Thermo Fisher); 1 μg RNA per sample was converted into cDNA.
A high-capacity cDNA reverse transcription kit (cat. no. 4368814; Life Technologies) was used for cDNA synthesis as recommended by the supplier. PCR was performed by the ABI Prism 7300 sequence detection system, using Life Technologies TaqMan gene expression assays (Table S2) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. All samples were run in triplicate, in a 20 μl reaction volume. Results were obtained as threshold cycle (C T ) values. Expression levels were calculated using the ΔC T method. The values were calculated as the mean values of three independent measurements, and the expression levels of mRNA in all samples were defined as a ratio to GAPDH expression. The average fold change compared with the control group was calculated using ΔΔC T method from RT-PCR data.
A high-capacity cDNA reverse transcription kit (cat. no. 4368814; Life Technologies) was used for cDNA synthesis as recommended by the supplier. PCR was performed by the ABI Prism 7300 sequence detection system, using Life Technologies TaqMan gene expression assays (Table S2) according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. All samples were run in triplicate, in a 20 μl reaction volume. Results were obtained as threshold cycle (C T ) values. Expression levels were calculated using the ΔC T method. The values were calculated as the mean values of three independent measurements, and the expression levels of mRNA in all samples were defined as a ratio to GAPDH expression. The average fold change compared with the control group was calculated using ΔΔC T method from RT-PCR data.
8
Validating HCC Gene Expression by RT-qPCR
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We carried out validation by quantitative reverse transcriptase (RT) real-time PCR for a selected group of genes (BIRC5 and SLC22A1 in the HCV group; CLEC1B in the HBV group; FGFR4, HSF1, RNF187, HSP90AB1, and HSPB1 in the group without viral infection; and HGF, COLEC10, and CYP17A as genes common to all groups) to validate our next-generation sequencing (NGS) results. These genes were selected based on their up- or downregulation in our HCC samples.
A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used to synthesize 2000 ng cDNA by incubation as follows: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 5 min. The amplification steps were performed using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) with the following thermocycler protocol: 95 °C for 10 min + (95 °C for 15 s; 60 °C for 1 min) for 40 cycles. The ABI PRISM 7300 Detection System (Applied Biosystems, Thermo Fisher Scientific) was used to analyze the relative expression all target genes normalized to b-actin. The expression levels of all target genes were related as fold changes 2−ΔΔCt. The primers were designed and synthesized by Kaneka Eurogentec S.A. Liège (Supplementary Table S2 ).
A High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used to synthesize 2000 ng cDNA by incubation as follows: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min, and 4 °C for 5 min. The amplification steps were performed using SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) with the following thermocycler protocol: 95 °C for 10 min + (95 °C for 15 s; 60 °C for 1 min) for 40 cycles. The ABI PRISM 7300 Detection System (Applied Biosystems, Thermo Fisher Scientific) was used to analyze the relative expression all target genes normalized to b-actin. The expression levels of all target genes were related as fold changes 2−ΔΔCt. The primers were designed and synthesized by Kaneka Eurogentec S.A. Liège (
9
Quantitative RT-PCR Analysis of Gene Expression
10
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from the tissues using TRIzol reagent (Life Technologies). Residual genomic DNA in the total RNA sample was eliminated by DNase I treatment (RNase-free recombinant DNase I, TaKaRa BIO). Quantitative RT-PCR analyses were performed using StepOnePlus (Applied Biosystems) along with a high capacity cDNA reverse transcription kit (Applied Biosystems) and Fast SYBR Green Master Mix (Applied Biosystems). Each reaction included 1 µg of total RNA as a template. The primers for quantitative RT-PCR are shown in Table 1 [7] (link), [10] (link), [15] (link). The reference control gene was virtually defined as the average of the threshold cycles (Ct) for SgPGK, SgEF1α and Sgβ-actin as reported in Vandesompele et al. (2002) [16] (link).
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