RNA was prepared from cultured cells using an RNeasy Mini Kit (Qiagen Japan, Tokyo, Japan), and first-strand cDNA was synthesized using a High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA), according to the instructions from the manufacturer. Total RNA (1 μg) was then reverse-transcribed. To validate gene expression changes, quantitative real-time reverse transcription PCR (RT-PCR) analysis was performed with a Prism 7500HT sequence detection system (Applied Biosystems) using Taq-Man gene expression assays for human CD46 (Hs00611257_m1), CD55 (Hs00167090_m1), CD59 (Hs00174141_m1), and 18S ribosomal RNA (4319413E) according to the specifications of the manufacturer (Applied Biosystems). Thermal cycler conditions were as follows: hold for 10 min at 95°C, followed by two-step PCR for 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were performed in duplicate. Amplification data were analyzed using Sequence Detection Software version 1.4.1 (Applied Biosystems), with 18S ribosomal RNA used as an endogenous control.
Taqman gene expression assays for human
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
TaqMan Gene Expression Assays are pre-designed, gene-specific assays for quantifying gene expression levels. They utilize TaqMan probe technology to provide accurate and reliable quantification of target gene transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression cell to ct kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
T100 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sds 1, by Thermo Fisher Scientific (1 mentions)
Abi 7500 fast real time pcr platform, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Experion automated electrophoresis station, by Bio-Rad (1 mentions)
Pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex system, by Thermo Fisher Scientific (1 mentions)
10 protocols using taqman gene expression assays for human
1
Quantitative RT-PCR Analysis of CD Markers
2
ABCB1 Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA isolation and cDNA synthesis was performed using the TaqMan Gene Expression Cell-to-CT Kit (Ambion) following the manufacturer’s protocol. Expression assays were performed using TaqMan Gene Expression Assays for human ABCB1 (Hs00184491_m1) and endogenous control human ACTB (Hs01060665_g1) with TaqMan Gene Expression Master Mix according to the manufacturer’s protocol (Applied Biosystems, Carlsbad, CA, USA). Reverse transcriptase reactions and real-time PCR were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). Briefly, the reactions were incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. In each experiment, triplicate reactions were performed per sample. Finally, the relative expression per sample was calculated as the ratio of ABCB1 to ACTB. Specifically, relative ABCB1 mRNA content per sample was calculated as 2−ΔΔCT, where ΔCT = (mean of triplicate CTABCB1 − mean of triplicate CTACTB) and ΔΔCT = (ΔCT – mean ΔCT of all the samples).
3
Investigating CYP1A1 Regulation in Human Keratinocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human keratinocytes (SIK 28) cells grown to confluence as previously described [89 (link)] in 6-well plates were incubated with DMSO (1% (v/v)), or 20 nM of TCDD, PZ, FICZ, or IND (in DMSO 1% (v/v)) in the absence or presence of the methanolic extract R4 (10 ppm) for 2 h. After incubation, cells were washed with PBS, total RNA isolated using TRIzol reagent (ThermoFisher Scientific, Waltham, MA, USA), and cDNA synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, San Francisco, CA, USA) with a Bio-Rad T100 Thermal Cycler. cDNAs were quantitated using Applied Biosystems Taqman gene expression assays for human CYP1A1 (ID: Hs00153120_m1), and the housekeeping gene β-glucuronidase (GUSB; ID: Hs99999908_m1) with an Applied Biosystems 7500 Fast Sequence Detection System (ThermoFisher Scientific) as previously described [38 (link),90 ]. CYP1A1 mRNA levels were normalized to those of GUSB, and values expressed relative to GUSB levels in DMSO-treated cells (set to a value of 1.0) as per the delta-delta Ct (2−ΔΔCT) method [91 (link)].
4
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was reverse transcribed into cDNA with a High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems ® , Monza, Italy). Semi-quantitative real-time PCR was performed using Taqman Gene Expression Assays for human ACTB (Hs99999903_m1), IL-1b (Hs01555410_m1), NLRP3 (Hs00366465_m1), CASP1 (Hs00354836_m1), SOD2 (Hs00167309_m1) and HemeOH (Hs01110250_m1) genes (Applied Biosystems ® Thermo Fisher, Monza, Italy) with the ABI 7500 Fast Real-Time PCR platform (Applied Biosystems ® Thermo Fisher, Monza, Italy). The PCR amplification cycle was "standard mode": after denaturation at 96 C for 10 min, 40 PCR cycles were performed composed by 15 s at 95 C and a final melting step for 1 min at 60 C. All samples were analysed in triplicate using the SDS 1.4 software (Applied Biosystems ® Thermo Fisher, Monza, Italy). Results were normalized to ACTB expression and then to untreated cells according to 2 ÀDDCt method [27] .
5
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from SH-SY5Y using the RNAqueous®-Micro Kit (Ambion®, Padova, Italy) and reverse transcribed into cDNA with a High Capacity cDNA Reverse Transcription Kit containing RNase Inhibitor (Applied Biosystems®, Monza, Italy). Semi-quantitative real-time PCR was performed using Taqman Gene Expression Assays for human ACTB (Hs99999903_m1) and HemeOH (Hs01110250_m1) genes (Applied Biosystems®, Monza, Italy) with the Applied Biosystems 7500 Fast Real-Time PCR System. PCR amplification cycle was as follows: after denaturation at 96°C for 10 min, 40 PCR cycles were performed composed by 15 sec at 95°C and a final melting step for 1 min at 60°C. All samples were analyzed in triplicate (for further details see Tricarico et al. [18] ).
6
Quantitative Analysis of TRP Channels in Cultured TG Neurons
7
RT-qPCR Analysis of Liver and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RT-qPCR, total RNA was isolated from macro-dissected FFPE liver tissue samples and treated LX2 cells. After homogenization, total RNA was purified using the PureLink FFPE Total RNS isolation kit (Life Technologies, Carlsbad CA, USA) for FFPE samples, and RNEasy Mini kit (Qiagen, Hilden, Germany) for cell samples according to the protocols provided by the manufacturers. The integrity of the total RNA was analyzed on the Experion Automated Electrophoresis Station (Bio-Rad Laboratories GmbH, Münich, Germany).
Total RNA reverse transcription and RT-qPCR from samples were done as detailed previously (42 (link)). RT-qPCR was accomplished by using TaqMan Gene Expression Assays for human: decorin (DCN, Assay ID: Hs00370383_m1, Life Technologies) and human smooth muscle actin (ACTA2, Assay ID: Hs.PT.56a21389192) according to the manufacturer's protocol. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (GAPDH, Assay ID: Hs.PT.39a22214836, Integrated DNA Technologies) and 18S RNA (Part No.:4319413E) were used as endogenous controls. All samples were run in duplicates. Results were obtained as threshold cycle values. Expression levels were determined by using the 2−ΔΔCT method.
Total RNA reverse transcription and RT-qPCR from samples were done as detailed previously (42 (link)). RT-qPCR was accomplished by using TaqMan Gene Expression Assays for human: decorin (DCN, Assay ID: Hs00370383_m1, Life Technologies) and human smooth muscle actin (ACTA2, Assay ID: Hs.PT.56a21389192) according to the manufacturer's protocol. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (GAPDH, Assay ID: Hs.PT.39a22214836, Integrated DNA Technologies) and 18S RNA (Part No.:4319413E) were used as endogenous controls. All samples were run in duplicates. Results were obtained as threshold cycle values. Expression levels were determined by using the 2−ΔΔCT method.
8
Quantifying mRNA Expression in RA Synovial Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets were assayed by real-time PCR for mRNA expression using TaqMan gene expression assays for human CD248 (Hs.00535586), human PDPN (Hs00366766_m1), and human 18S (Hs.03928985) (Life Technologies). Samples were reverse transcribed using a Cells-to-cDNA kitorkit or TaqMan Reverse Transcription kit (both Life Technologies) with PCR Master Mix and TaqMan Universal Master Mix II reagent (Life Technologies). Assays were run on a 7900HT Real-Time PCR system (Life Technologies) according to the manufacturer’s protocol. Data were obtained as Ct values. Expression values for the gene of interest were normalized to those for 18S ribosomal RNA and were transformed to assume a doubling of product with each PCR cycle, calculated using the comparative threshold cycle (Ct) method and then 2–delta Ct formula, as the Ct gene of interest minus the Ct housekeeping gene. Results were expressed as the fold-change relative to values in the control, untreated samples. Each experiment was performed in duplicate and repeated in five different primary RA synovial fibroblast donor cell lines.
9
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cell lysate of HLMVECs grown in flow chamber slides using the Cytiva illustra RNAspin Mini Isolation Kit according to manufacturer protocol (Thermo Fisher Scientific). RNA was converted to cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific), and cDNA underwent PCR using TaqMan gene expression assays for human ANG2 (assay ID Hs00169867_m1), FOXO1 (Hs00231106_m1), and KLF2 (Hs00360439_g1) mRNA (Thermo Fisher Scientific). PCR reactions were quantified using a QuantStudio 6 Flex System from Applied Biosystems, Thermo Fisher Scientific, and gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression (Hs02758991_g1, Thermo Fisher Scientific) using the 2-ΔΔCT method.
10
TPIT Expression in Neuroendocrine Neoplasms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was obtained from three representative formalin-xed and para n-embedded sections (FFPE, 8μm) for each sample. Total RNA was extracted from FFPE tissues after microdissection by using Maxwell® RNA FFPE Kit and Maxwell 16 system (Promega, Madison, USA) according to the recommendations of the manufacturer. RNA was quanti ed using Qubit™ RNA XR Assay Kit (Invitrogen -Thermo Fisher Scienti c, Whaltam, USA). Reverse transcription (RT) was carried out on 800ng of total RNA using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c, Whaltam, USA) according to the manufacturer's protocol.
The mRNAs expression levels of TPIT in neuroendocrine neoplasms and control hypophysis samples were determined by quantitative (q) PCR by using a Quantstudio 6/7 real-time PCR system (Thermo sher). qPCR reactions were prepared using reverse-transcribed RNA, TaqMan Gene Expression Assays for human TPIT (ThermoFisher, Hs00193027) and Beta-2 microglobulin (ThermoFisher, Hs00187842), and 1× TaqMan universal PCR Mastermix (Thermo Fisher scienti c). TPIT expression was normalized to Beta-2 microglobulin expression and quanti ed using the ΔΔCT method.
The mRNAs expression levels of TPIT in neuroendocrine neoplasms and control hypophysis samples were determined by quantitative (q) PCR by using a Quantstudio 6/7 real-time PCR system (Thermo sher). qPCR reactions were prepared using reverse-transcribed RNA, TaqMan Gene Expression Assays for human TPIT (ThermoFisher, Hs00193027) and Beta-2 microglobulin (ThermoFisher, Hs00187842), and 1× TaqMan universal PCR Mastermix (Thermo Fisher scienti c). TPIT expression was normalized to Beta-2 microglobulin expression and quanti ed using the ΔΔCT method.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!