Total RNA was isolated using RNeasy Mini Kit (Qiagen) and treated with DNase I according to the manufacturer’s instructions. qRT-PCR was performed with the QuantiTect Probe RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems). Target gene expression was normalized to GAPDH mRNA, which was quantified using TaqMan human GAPDH control reagents (Applied Biosystems). Ct values of greater than 35 were regarded as “not detected” (“N.D.”). The sequences of the primers and probes used in the present study are listed in Table S1 . Oligonucleotides were obtained from Sigma-Aldrich.
Taqman human gapdh control reagents
Manufactured by Thermo Fisher Scientific
The TaqMan human GAPDH control reagents are a set of tools used in qPCR (quantitative Polymerase Chain Reaction) experiments. The GAPDH gene is a commonly used reference gene for normalization in gene expression analysis. The control reagents provide a reliable and consistent positive control for the detection of the GAPDH gene, allowing researchers to ensure the accuracy and integrity of their qPCR results.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (4 mentions)
Quantitect probe one step rt pcr kit, by Qiagen (4 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Quantitect probe rt pcr kit, by Qiagen (2 mentions)
Eukaryotic 18s rrna endogenous control, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman ribosomal rna control reagent, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Big dye 1.1 sequencing kit, by Thermo Fisher Scientific (1 mentions)
Platinum taq polymerase, by Thermo Fisher Scientific (1 mentions)
7 protocols using taqman human gapdh control reagents
1
Quantitative RT-PCR for Gene Expression
2
Quantitative RT-PCR for Gene Expression
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Total RNA was isolated as described above. qRT-PCR was performed with the QuantiTect Probe One-Step RT-PCR Kit (Qiagen) on a StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels of target genes were normalized to those of the GAPDH transcript or 18S rRNA, which were quantified using TaqMan human GAPDH control reagents (Applied Biosystems) or eukaryotic 18S rRNA endogenous control (Applied Biosystems), respectively. Probes and primers were obtained from Sigma-Aldrich. The sequences of the primers and probes used in the present study are listed in Supplementary Table 3 .
3
Quantifying hTERT Expression in ASC-Spiked ADSCs
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Total RNA was isolated from ASC52telo cells spiked into ADSCs using an RNeasy Mini Kit with DNase I treatment (QIAGEN) according to the manufacturer's instructions. RNA concentration was measured using the NanoDrop ND-1000 (Thermo Fisher Scientific). qRT-PCR was performed with a QuantiTect Probe RT-PCR Kit (QIAGEN) on a 7300 Real-Time PCR System (Applied Biosystems). The PCR condition was as follows. After initial incubations at 50 °C for 30 min and 95 °C for 15 min, 40 cycles of amplification were carried out at 95 °C for 15 s and 60 °C for 1 min. Total RNA (5 ng per sample) was used for analysis. The levels of hTERT expression were normalized by those of GAPDH expression, which were quantified using TaqMan human GAPDH control reagents (Applied Biosystems). Primers and probes incorporating 5′-FAM reporter dye and 3′-TAMRA quencher dye for qRT-PCR were obtained from Sigma–Aldrich. The primer and probe sequences targeting hTERT gene were as follows: 5′-CCTGTTTCTGGATTTGCAGGTG-3′ (forward primer), 5′-GCACACATGCGTGAAACCTG-3′ (reverse primer), and 5′-CAGCCTCCAGACGGTGTGCACCAAC-3′ (probe).
4
Quantitative RNA Expression Analysis
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Total RNA was isolated from cells using an RNeasy Mini Kit with DNase I treatment (Qiagen), according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). In the spike experiments, 253G1 cells and primary human cardiomyocytes (PromoCell) were mixed at a defined cell number before RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems). Total RNA (50 ng per sample) was used for the analysis. The expression levels of target genes were normalized to those of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript or ribosomal RNA, which were quantified using TaqMan human GAPDH control reagents (Life Technologies) or TaqMan ribosomal RNA control reagents (Life Technologies). Probes labeled with 5′-FAM/3′-TAMRA and primers were obtained from Sigma–Aldrich. The sequences of primers and probes used in the present study are listed in Table S1 . All qRT-PCR reactions were run at 45 cycles.
5
qRT-PCR for Gene Expression Analysis
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Total RNA was treated with DNase I and isolated using RNeasy Mini Kit (Qiagen Hilden, Germany) according to the manufacturer's instructions. Quantitative RT-PCR was performed using the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on StepOnePlus Real Time PCR system (Life Technologies). Gene expression levels were normalized to GAPDH expression levels, which were quantified using TaqMan human GAPDH control reagents (Life Technologies). Primers and probes were obtained from Sigma-Aldrich. The sequences of primers and probes are listed in Table S1 .
6
Detection of Common EML4-ALK Variants
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At thromboDx, quantitative PCR Taqman assays from Life Technologies (Hs03654556, Hs03654557, and Hs03654558) were used for detection of the three most common EML4-ALK variants (v1 [e13:a20], v2 [e20:a20], and v3 [e6:a20], with breakpoints at EML4 exons 13, 20, and 6 and at ALK exon 20, respectively), according to the manufacturer's instructions. Quantitative PCR was run on the ABI7500 (Applied Biosystems, Foster City, CA) for 60 cycles in a standard TaqMan program with TaqMan human GAPDH control reagents (Life Technologies, 402869) as input control. EML4-ALK breakpoints were confirmed by Sanger sequencing of RT-PCR products, using the Big-Dye 1.1 sequencing kit (Applied Biosystems). At Pangaea Biotech, EML4-ALK was amplified by PCR and visualized in agarose gels. RNA was converted to cDNA by superscript III (Life Technologies) and amplified using 40 ng of cDNA and 1U Platinum Taq polymerase (Invitrogen) in a 20 μl reaction. Specific forward and reverse primers were used for the three most common EML4-ALK variants (Supplementary Table S5 ). EML4-ALK breakpoints were confirmed by Sanger sequencing of RT-PCR products, using the Big-Dye 1.1 sequencing kit (Applied Biosystems).
7
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. qRT-PCR was performed using the QuantiTect Probe One-step RT-PCR Kit (Qiagen) on the StepOnePlus Real Time PCR System (Life Technologies, Carlsbad, CA, USA). Gene expression levels were normalized to GAPDH expression levels, which were quantified using TaqMan Human GAPDH Control Reagents (Life Technologies). Primers and probes were obtained from Sigma-Aldrich. The sequences of primers and probes are listed in Table S1 .
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