Quantitech primer assay

Manufactured by Qiagen

Sourced in Sweden

QuantiTech primer assays are a set of pre-designed and validated primer pairs for use in quantitative PCR (qPCR) experiments. These primer assays target specific gene sequences and are optimized for reliable and sensitive gene expression analysis.

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QuantiTech primers (9 citations)

24 protocols using quantitech primer assay

Quantifying Gene Expression in Stem Cells

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RNA was extracted from hiPSC and hESCs growing in a culture dish to 80 % of confluence, using RNeasy Plus Mini Kit (QIAGEN, 74134). RNA purity and concentration were confirmed using NanoDrop 8000 (Thermo Scientific).
RT-qPCR was performed on a Mastercycler ep RealPlex² Real Time PCR ThermoCycler (Eppendorf, 2894) using Verso SYBR Green 1-Step qRT-PCR Low ROX Kit (Thermo Scientific, AB-4106). Reactions were set up according to the manual using 1 ng RNA, 2.5 μL primers, 0.25 μL Verso Enzyme Mix, 1X SYBR mix, 1.25 μL RT Enhancer, and nuclease-free water for a total volume of 25 μL. The 1-Step RT-qPCR thermal cycling program was carried out at 50 °C for 15 min; 95 °C for 15 min; and 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, with subsequent melting curve analysis. The primers used for RT-qPCR were Hs_FMR1_1_SG (QIAGEN QuantiTech^® Primer Assay, QT00017479) and Hs_GAPDH_1_SG (QIAGEN QuantiTech^® Primer Assay, QT00079247). Expression of transcripts of target genes was normalized to Gapdh.

Hiroi N., Brown J.R., Haile C.N., Ye H., Greenberg M.E, & Nestler E.J. (1997). FosB mutant mice: Loss of chronic cocaine induction of Fos-related proteins and heightened sensitivity to cocaine’s psychomotor and rewarding effects. Proceedings of the National Academy of Sciences of the United States of America, 94(19), 10397-10402.

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Quantification of RNA Expression in Colon Cancer Cells

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Total RNA was extracted from CaCo-2 and HT-29 cells using the RNeasy mini kit (Qiagen, Sollentuna, Sweden). RNA concentration and purity were measured using a NanoDrop ND-1000 spectrophotometer (Saveen Werner AB, Limhamn, Sweden). First, 1.5 μg of total RNA was treated with DNase then converted to cDNA using the Superscript III First Strand synthesis system (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Commercially available primers (Quantitech Primer Assay, Qiagen, Sollentuna, Sweden) were used in combination with RT² real-time™ SYBR Green/ROX Polymerase chain reaction (PCR) master mix (Qiagen, Sollentuna, Sweden) to quantify the mRNA expression of IFNLR1, IL-10RB, IFNλ1, IFNλ2, PKR, OAS-2, MxA, ISG15, iNOS, TLR3, MDA5, RIG-I, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The same cDNA was used to examine IFNβ using commercially available Taqman primers and the Taqman mastermix (Thermo Fisher Scientific, Stockholm, Sweden). Real-time PCR was performed using an ABI Prism 7500 Sequence detecting system (Thermo Fisher Scientific, Stockholm, Sweden). Gene expression levels were normalized to the expression of GAPDH (Ct). The data are presented as 2^−ΔCt. In all experiments, a Ct value of ≥35 was considered to be below the detection level and thus genes with a Ct value of greater than 35 were deemed to be not expressed.

Stone V.M., Ringqvist E.E., Larsson P.G., Domsgen E., Holmlund U., Sverremark-Ekström E, & Flodström-Tullberg M. (2021). Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells—A Strategy Used by Coxsackie B Virus to Evade the Host’s Innate Immune Response at the Primary Site of Infection?. Microorganisms, 9(1), 105.

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Quantitative mRNA Expression Analysis

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Total mRNA was isolated from the prefrontal cortex and hippocampus using the miRNeasy mini kit (Qiagen Inc.) according to the manufacturer’s recommendations. Total RNA concentration and purity were measured by a Nanodrop (Thermo Scientific) at 235, 260 and 280 nm. cDNA was prepared from 1ug RNA in a 20-μL reaction using QuantiTect Reverse Transcription Kit (Qiagen). Each PCR (20 μL), containing 2 μL cDNA (12 ng), 10 μL Quanti Fast SYBR Green PCR Master Mix (Qiagen) and 2 μL PCR primer (QuantiTech Primer Assay, Qiagen), was run on a LightCycler 480 (Roche, Sweden). The following primers were used: Efna3 QuantiTech Primer Assay (QT00320026), Epha4 QuantiTech Primer Assay (QT00093576), Dlg4 QuantiTech Primer Assay (QT00121695) and Syp QuantiTech Primer Assay (QT01042314), all from Qiagen. Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplified transcripts were quantified with the relative standard curve and normalized by the cDNA concentration using the Quant-IT OliGreen ssDNA Assay kit (Fisher Scientific).

Shiadeh S.M., Goretta F., Svedin P., Jansson T., Mallard C, & Ardalan M. (2024). Long-term impact of maternal obesity on the gliovascular unit and ephrin signaling in the hippocampus of adult offspring. Journal of Neuroinflammation, 21, 39.

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Validation of gene expression by qPCR

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Sybr green real-time qPCR was used to validate the array gene expression results (DyNAmo SYBR Green qPCR Kit with ROX) using primers designed by Qiagen (Qiagen QuantiTech primer assay). The house keeping gene GAPDH was used as an endogenous control alongside the Vector, no E2 expressing U20S cell line to normalize the results using the ΔΔct method. A list of genes validated can be found in Table S1.

Gauson E.J., Windle B., Donaldson M.M., Caffarel M.M., Dornan E.S., Coleman N., Herzyk P., Henderson S.C., Wang X, & Morgan I.M. (2014). Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein. Virology, 468-470, 10-18.

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Validating E2-Regulated Genes via qPCR

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To validate the genes regulated by E2-WT and E2-Brd4 expressing U20S clones, a Sybr green protocol was followed (DyNAmo SYBR Green qPCR Kit with ROX, Cat no: F-400RL), using primers designed by Qiagen, (Qiagen QuantiTech primer assay). Results were all normalized to a GAPDH enogenous control, and then further normalized to VEC 1:1 (non-E2 control), which was made 1. The ΔDct method was used for this validation.

Gauson E.J., Wang X., Dornan E.S., Herzyk P., Bristol M, & Morgan I.M. (2015). Failure to interact with Brd4 alters the ability of HPV16 E2 to regulate host genome expression and cellular movement. Virus research, 211, 1-8.

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Quantifying Gene Expression in Rat Liver

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Total RNA was extracted from frozen liver (~ 20 mg) using a RNeasy Plus Universal Mini Kit (QIAGEN), as per manufacturer’s instructions. The concentration of eluted RNA was measured using a Nanodrop DN-1000 spectrophotometer. cDNA synthesis was carried out using the qScript synthesis kit, following the manufacturer’s protocol (Quantabio). mRNA expression was measured by quantitative (Q)-PCR using SYBR Green Mastermix (Eurogentec Ltd.) and the DNA Engine Opticon 2 system (BioRad). Primers were obtained from QuantiTech Primer Assay (QIAGEN) and product details are as follows: Rn_Hk2_1 QT00190764, Rn_Pfkl_1 QT00175651, Rn_Ldha_2 QT02336243, Rn_Higd1a_1 QT00372428, Rn_Stoml2_1 QT01571724 , Rn_Slc25a11_1 QT01082914 , Rn_Actb_1 QT00193473. In the instance of Cox7a2l, QuantiFast SYBR Green PCR kit (QIAGEN) and QuantStudio 1 Real-Time PCR System (Applied Biosystems, Thermo Fisher) were used. The primer was obtained from QuantiTect Primer Assays (QIAGEN) with the following product details: Rn_Cox7a2l_1_SG. In all cases, transcript levels were normalised to levels of Actb and fold change determined using the 2^−ΔΔCT method, with expression in vehicle/normoxic animals normalised to 1.

O’Brien K.A., McNally B.D., Sowton A.P., Murgia A., Armitage J., Thomas L.W., Krause F.N., Maddalena L.A., Francis I., Kavanagh S., Williams D.P., Ashcroft M., Griffin J.L., Lyon J.J, & Murray A.J. (2021). Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress. BMC Biology, 19, 265.

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Optimized Noggin Expression Analysis

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RNA extraction was performed by using a Maxwell 16 LEV simplyRNA Tissue Kit (Promega, Madison, WI) followed by RNA quantification with a NanoDrop 2000 (Thermo Scientific, Wilmington, DE). 50 ng of sample RNA was immediately reverse transcribed into cDNA with an Omniscript RT kit according to the manufacturer’s instructions (Qiagen, Venlo, Netherlands) and frozen at -20° C until analyzed by qPCR. cDNA samples were mixed with QuantiTech SYBR Green PCR kit (Qiagen, Venlo, Netherlands) and appropriate primers (QuantiTech Primer Assay, Qiagen, Venlo, Netherlands) for mouse noggin (QT00256585), beta-Actin (QT00095242), or GAPDH (QT01658692). Quantative PCR was performed on a Bio-Rad iCycler (Bio Rad, Hercules, CA) for 40 cycles. Data were analyzed with Biogazelle QBase+ software (Zwijnaarde, Belgium). For the dose response of noggin expression to increasing rhBMP-2 dose (section 2.8) noggin expression was normalized to MC3T3-E1 cells that had not received rhBMP-2 (0 μg/mL). For experiments to determine levels of knockdown after treatment with siRNA in cells that had been exposed to 10 μg/mL of rhBMP-2, noggin expression was normalized to MC3T3-E1 cells that were exposed to 10 μg/mL of rhBMP-2 but were not treated with siRNA.

Kowalczewski C.J, & Saul J.M. (2015). Surface-Mediated Delivery of siRNA from Fibrin Hydrogels for Knockdown of the BMP-2 Binding Antagonist Noggin. Acta biomaterialia, 25, 109-120.

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Hippocampal Gene Expression Analysis

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At P12 and P45 (n = 10 per treatment group per sex), mice were deeply anaesthetized via intraperitoneal administration of pentobarbital (Pentacour), and the right and left hippocampi were rapidly dissected and stored at −80°C until analysis. Total mRNA was isolated from the hippocampus using the miRNeasy mini kit (Qiagen Inc.) according to the manufacturer's recommendations. Total RNA concentration and purity were measured by a Nanodrop (Thermo Scientific) at 235, 260, and 280 nm. cDNA was prepared from 1ug RNA in a 20‐μl reaction using QuantiTect Reverse Transcription Kit (Qiagen). Each PCR (20 μl), contained 2‐μl cDNA (12 ng), 10‐μl Quanti Fast SYBR Green PCR Master Mix (Qiagen), and 2‐μl PCR primer (QuantiTech Primer Assay, Qiagen), were run on a LightCycler 480 (Roche, Sweden). The following primers were used Nlgn1 QuantiTech Primer Assay (QT00167580), Nrxn3 QuantiTech Primer Assay (QT00166621), and Pvrl1 (nectin‐1) QuantiTech Primer Assay (QT00171703) all from Qiagen. Melting curve analysis was performed to ensure that only one PCR product was obtained. For quantification and for estimation amplification efficiency, a standard curve was generated using increasing concentrations of cDNA. The amplified transcripts were quantified with the relative standard curve and normalized by the cDNA concentration using the Quant‐IT OliGreen ssDNA Assay kit (Fisher Scientific).

Ardalan M., Chumak T., Quist A., Hermans E., Hoseinpoor Rafati A., Gravina G., Jabbari Shiadeh S.M., Svedin P., Alabaf S., Hansen B., Wegener G., Westberg L, & Mallard C. (2022). Reelin cells and sex‐dependent synaptopathology in autism following postnatal immune activation. British Journal of Pharmacology, 179(17), 4400-4422.

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Quantitative Analysis of Insulin Transcripts

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Expression of insulin transcripts was detected with RT-qPCR. For cDNA synthesis, 300 ng of total RNA was reverse transcribed with SuperScript VI, Random Hexamers, dNTP, RNaseOUT RNase Inhibitor (all from Invitrogen), as recommended by the manufacturer. With cDNA input equivalent to 4 ng total RNA, using Power SYBR Green PCR Master Mix (Applied Biosystems) with primers F: 5’-GAAGCGTGGCATTGTGGAA-3’ and R: 5’- GCGTCTAGTTGCAGTAGTTCT-3’. The PCR programme (10 min at 95°C, 30/34× (15s 95°C, 1min 62°C)) was run on Mastercycler ×50 (Eppendorf, Hamburg, Germany). For the INS-IGF2 transcript 2, we used QuantiTech Primer Assay (Qiagen) using PCR conditions as described for insulin. In all reactions, normal pancreas and normal adrenal gland were used as controls.
PCR products were separated by electrophoresis on 2% TAE/agarose gels using UltraPure Agarose (Invitrogen). Pictures were acquired and quantified by fluorescence using the Gel-doc XR+ system and ImageLab 5.2.1 software (both from Bio-Rad).
Relative quantification of insulin expression was also performed by qPCR, by comparing the Ct value from patient samples with Ct values from a standard curve made from dilutions of the insulin cDNA from the pancreas. The reaction was run in a 384 format on QuantStudio7 (Applied Biosystems), otherwise as described for regular PCR.

Følling I., Wennerstrøm A.B., Eide T.J, & Nilsen H.L. (2021). Phaeochromocytomas overexpress insulin transcript and produce insulin. Endocrine Connections, 10(8), 815-824.

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Efficient qRT-PCR Quantification Protocol

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Detailed methods used for qRT-PCR have been described previously (28 (link)). Specific primers were purchased from Qiagen (QuantiTech Primer Assays), for each primer set the efficiency was > 95% and a single product was seen on melt curve analysis. Relative expression levels were calculated using the 2_-ΔΔCt method normalizing to GAPDH expression levels for each treatment and the fold increase in expression was relative to the smallest expression level.

Compan V., Martín-Sánchez F., Baroja-Mazo A., López-Castejón G., Gomez A.I., Verkhratsky A., Brough D, & Pelegrín P. (2014). Apoptosis-associated speck-like protein containing CARD forms specks but does not activate caspase-1 in the absence of NLRP3 during macrophage swelling. Journal of immunology (Baltimore, Md. : 1950), 194(3), 1261-1273.

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