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Taqman pcr universal mastermix

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TaqMan PCR Universal Mastermix is a ready-to-use reagent that contains all the necessary components for performing real-time quantitative PCR (qPCR) reactions, including DNA polymerase, dNTPs, buffer, and passive reference dye. It is designed to be used with TaqMan probe-based detection, enabling sensitive and specific gene expression analysis.

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Lab products found in correlation

High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (12 mentions) Rneasy mini kit, by Qiagen (3 mentions) Trizol reagent, by Thermo Fisher Scientific (3 mentions) Sybr green pcr universal mastermix, by Thermo Fisher Scientific (2 mentions) β actin, by Thermo Fisher Scientific (2 mentions) Quantitect reverse transcription kit, by Qiagen (2 mentions) Bax rn02532082 g1, by Thermo Fisher Scientific (2 mentions) Bcl 2 rn99999125 m1, by Thermo Fisher Scientific (2 mentions) Caspase 3 rn00563902 m1, by Thermo Fisher Scientific (2 mentions) Abi prism 7000, by Thermo Fisher Scientific (2 mentions) Rneasy micro kit, by Qiagen (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Ab7900 machine, by Thermo Fisher Scientific (1 mentions) Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions) Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) High capacity cdna archive kit, by Thermo Fisher Scientific (1 mentions) Rneasy kit, by Qiagen (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)

13 protocols using taqman pcr universal mastermix

1

Kidney Gene Expression Analysis

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Total RNA was extracted from kidney tissue using Trizol. cDNA was synthesised using Superscript III (Life Technologies) first strand synthesis. The cDNA was subjected to standard curve measurement to ensure efficiency prior to real time PCR, which was done using SYBR green (Life Technologies, Australia) for MCP-1, fibronectin, TGFβ, collagen IV, GLUT1 and GLUT2 using actin as the endogenous control. PCR for SGLT2 and SGLT1 were done using Taqman PCR Universal Mastermix (Applied Biosystems). The RTPCR was performed on the AB7900 machine (Applied Biosystems, Australia). Gene expression was quantified relative to actin. The primers are listed in Table 2.
Gangadharan Komala M., Gross S., Mudaliar H., Huang C., Pegg K., Mather A., Shen S., Pollock C.A, & Panchapakesan U. (2014). Inhibition of Kidney Proximal Tubular Glucose Reabsorption Does Not Prevent against Diabetic Nephropathy in Type 1 Diabetic eNOS Knockout Mice. PLoS ONE, 9(11), e108994.
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2

Quantitative RT-PCR Analysis of Annexin A1

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Total cellular RNA was isolated using the RNeasy Mini Kit according to the manufacturer's protocol (Qiagen) and first strand cDNA was synthesized with an oligo (dT) primer and a SuperScript™ First Strand Synthesis System (Invitrogen, Rockville, Maryland, USA). Quantitative real-time PCR analysis was conducted by using TaqMan PCR Universal Mastermix (for β-actin) or SYBR GREEN PCR Master Mix (for Annexin A1; Applied Biosystems, Darmstadt, Germany). Quantification was performed by the comparative ΔCT method normalizing CT-values to β-actin.
Rohwer N., Bindel F., Grimm C., Lin S.J., Wappler J., Klinger B., Blüthgen N., Du Bois I., Schmeck B., Lehrach H., de Graauw M., Goncalves E., Saez-Rodriguez J., Tan P., Grabsch H.I., Prigione A., Kempa S, & Cramer T. (2015). Annexin A1 sustains tumor metabolism and cellular proliferation upon stable loss of HIF1A. Oncotarget, 7(6), 6693-6710.
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3

Quantitative Analysis of Progerin and Lamin C

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Collected cells or tissues were homogenized in TRIzol reagent (Life Technologies) and RNA was extracted with the RNeasy Mini kit following the manufacturer’s instructions (QIAGEN). cDNA was synthesized with the QuantiTect Reverse Transcription kit (QIAGEN) using 1 μg of total RNA, and then RT-qPCR analysis of mouse tissues was performed. For progerin analysis, TaqMan PCR Universal Mastermix (Applied Biosystems) and the following oligonucleotides and probe were used: MmProgerin_fwd (5’-TGAGTACAACCTGCGCTCAC-3’), MmProgerin_rev (5’-TGGCAGGTCCCAGATTACAT-3’) and MmProgerin_probe (5’-CGGGAGCCCAGAGCTCCCAGAA-3’); using a β-actin (Applied Biosystems) as endogenous control. For lamin C analysis, we used SYBR green PCR Universal Mastermix (Applied Biosystems) and the oligonucleotides Lmnc_fwd (5’-CGACGAGGATGGAGAAGAGC-3’) and Lmnc_rev (5’-AGACTTTGGCATGGAGGTGG-3’) for lamin C; or Actb_Fwd (5’-CTGAGGAGCACCCTGTGCT-3’) and Actb_Rev (5’-GTTGAAGGTCTCAAACATGATCTG-3’) for β-actin as endogenous control.
Santiago-Fernández O., Osorio F.G., Quesada V., Rodríguez F., Basso S., Maeso D., Rolas L., Barkaway A., Nourshargh S., Folgueras A.R., Freije J.M, & López-Otín C. (2019). Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome. Nature medicine, 25(3), 423-426.
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4

Gene Expression Analysis of Fetal Mouse Lung

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Total RNA was isolated from E18.5 lung tissue (n = 12) and purified with the RNeasy kit (Qiagen, Valencia, CA). RNA was reverse transcribed with the High Capacity cDNA Archive Kit with random primers (Applied Biosystems, Foster City, CA). RT-PCR was performed using commercially available sense and antisense oligonucleotides (Applied Biosystems), TaqMan PCR Universal Master Mix (Applied Biosystems), and gene specific TaqMan probes. The Applied Biosystems PRISM 7900HT Sequence Detection System (Applied Biosystems) was used to detect amplification level. Glyceraldehyde-3-phosphate dehydrogenase was used as endogenous control. Relative gene expression was quantitated by the 2−ΔΔCT method.23 (link) The genes studied were corticotropin-releasing hormone (Crh), corticotropin-releasing hormone receptor 1 (Crhr1), corticotropin-releasing hormone receptor 2 (Crhr2), insulin-like growth factor 1 (Igf1), insulin-like growth factor 2 (Igf2), surfactant protein A1 (Sftpa1), surfactant protein B (Sftpb), surfactant protein C (Sftpc), short stature homeobox 2 (Shox2), thyrotropin-releasing hormone (Trh), and urocortin 2 (Ucn2).
Pew B.K., Harris R.A., Sbrana E., Guaman M.C., Shope C., Chen R., Meloche S, & Aagaard K. (2016). Structural and transcriptomic response to antenatal corticosteroids in an Erk3-null mouse model of respiratory distress. American journal of obstetrics and gynecology, 215(3), 384.e1-384.e89.
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5

Quantitative Analysis of Progerin and Lamin C

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Collected cells or tissues were homogenized in TRIzol reagent (Life Technologies) and RNA was extracted with the RNeasy Mini kit following the manufacturer’s instructions (QIAGEN). cDNA was synthesized with the QuantiTect Reverse Transcription kit (QIAGEN) using 1 μg of total RNA, and then RT-qPCR analysis of mouse tissues was performed. For progerin analysis, TaqMan PCR Universal Mastermix (Applied Biosystems) and the following oligonucleotides and probe were used: MmProgerin_fwd (5’-TGAGTACAACCTGCGCTCAC-3’), MmProgerin_rev (5’-TGGCAGGTCCCAGATTACAT-3’) and MmProgerin_probe (5’-CGGGAGCCCAGAGCTCCCAGAA-3’); using a β-actin (Applied Biosystems) as endogenous control. For lamin C analysis, we used SYBR green PCR Universal Mastermix (Applied Biosystems) and the oligonucleotides Lmnc_fwd (5’-CGACGAGGATGGAGAAGAGC-3’) and Lmnc_rev (5’-AGACTTTGGCATGGAGGTGG-3’) for lamin C; or Actb_Fwd (5’-CTGAGGAGCACCCTGTGCT-3’) and Actb_Rev (5’-GTTGAAGGTCTCAAACATGATCTG-3’) for β-actin as endogenous control.
Santiago-Fernández O., Osorio F.G., Quesada V., Rodríguez F., Basso S., Maeso D., Rolas L., Barkaway A., Nourshargh S., Folgueras A.R., Freije J.M, & López-Otín C. (2019). Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome. Nature medicine, 25(3), 423-426.
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6

Gene Expression Analysis of Inflammatory Markers

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RNA was extracted from cells with TRI Reagent (Molecular Research Center). Complementary DNA (cDNA) was synthesized from 1 mg of total RNA (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). The relative amount of mRNA was measured by real-time PCR assay with 1 ml of cDNA and TaqMan PCR Universal Master Mix (Applied Biosystems). Human TaqMan gene expression probes included IL23A, IL12B, ICAM1, VCAM1, PECAM1, CXCL9, CXCL10, CXCL11, IL6, and GAPDH (Applied Biosystems). The PCR conditions used were those recommended by the manufacturer.
Espígol-Frigolé G., Planas-Rigol E., Ohnuki H., Salvucci O., Kwak H., Ravichandran S., Luke B., Cid M.C, & Tosato G. (2016). Identification of IL-23p19 as an endothelial proinflammatory peptide that promotes gp130-STAT3 signaling. Science signaling, 9(419), ra28.
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7

Quantitative RT-PCR Analysis of Glioma Markers

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Total RNA was extracted from glioma tumor tissues and adjacent nontumorous tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) was reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). RT-PCR was performed with TaqMan® PCR Universal Master Mix (Applied Biosystems) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. GAPDH acted as an internal control. The primer sequences were listed as follows: CTGF, 5′-GTGGATGACAAGTCCAGGCG-3′ (forward) and 5′-GTCCTCCTCTTGTTCTTGTGC-3′ (reverse); TGF-β1, 5′-ACCTGTCGACAAGACCACATG-3′ (forward) and 5′-CGAGGTTTGGACCTTACAGGAT-3′ (reverse); collagen I, 5′-ACGTCCTGGTGAAGTTGGTC-3′ (forward) and 5′-CAGGGAAGCCTCTTTCTCCT-3′ (reverse); collagen IIII, 5′-TGGCCCTGACCCAACTATGAT-3′ (forward) and 5′-GCACTTTTTGCCCTTCTTAATGTT-3′ (reverse); αSMA, 5′-AGAATCAGAGTCAGGCGTCC-3′ (forward) and 5′-AGTAGAAGGCTGTCACCAAGAC-3′ (reverse); GAPDH, 5′-ATGACATCAAGAAGGTGGTG-3′ (forward) and 5′-CATACCAGGAAATGAGCTTG-3′ (reverse). Relative expression level was calculated with the 2−ΔΔCt method.
Sun Y., Jin J.G., Mi W.Y., H.a.o.-.W.u., Zhang S.R., Meng Q, & Zhang S.T. (2018). Long Noncoding RNA UCA1 Targets miR-122 to Promote Proliferation, Migration, and Invasion of Glioma Cells. Oncology Research, 26(1), 103-110.
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8

Retinal Ischemia-Reperfusion Gene Expression

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From retinal tissue harvested 24 h after ischemia, total RNA from ¼ of retina was extracted using a column-purification based kit (RNeasy Micro Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Darmstadt, Germany). Real time polymerase chain reactions (RT-PCR) were done with TaqMan probe-based detection kit (TaqMan PCR universal mastermix, Applied Biosystems, Darmstadt, Germany). Following primers were used: Bax #Rn02532082_g1, Bcl-2 #Rn99999125_m1, Caspase-3 #Rn00563902_m1, NF-κB #Rn01399565_m1 (all from Applied Biosystems, Darmstadt, Germany). The PCR assays were then performed on a RT-PCR System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Reaction specificity was confirmed by running appropriate negative controls. Cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT). Relative gene expression in I/R injured retinal tissue either with Argon or room air was calculated in relation to the corresponding gene expression in the non-injured retinal tissue of each individual animal (ΔΔCT).
Ulbrich F., Schallner N., Coburn M., Loop T., Lagrèze W.A., Biermann J, & Goebel U. (2014). Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats. PLoS ONE, 9(12), e115984.
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9

Retinal Gene Expression after Ischemia

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From retinal tissue harvested 24 h and 48 h after ischemia, total RNA from ¼ of retina was extracted using a column-purification based kit (RNeasy Micro Kit, Qiagen, Hilden, Germany) according to the manufacturer´s instructions. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Darmstadt, Germany). Real time polymerase chain reactions (RT-PCR) were done with TaqMan® probe-based detection kit (TaqMan® PCR universal mastermix, Applied Biosystems, Darmstadt, Germany). Following primers were used: Caspase-3 #Rn00563902_m1, Bax #Rn02532082_g1 and Bcl-2 #Rn99999125_m1 (all from Applied Biosystems, Darmstadt, Germany). The PCR assays were then performed on a RT-PCR System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Reaction specificity was confirmed by running appropriate negative controls. Cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT). Relative gene expression in IR injured retinal tissue either with injection of ALF-186 or PBS was calculated in relation to the corresponding gene expression in the non-injured retinal tissue of each individual animal (ΔΔCT).
Ulbrich F., Kaufmann K.B., Meske A., Lagrèze W.A., Augustynik M., Buerkle H., Ramao C.C., Biermann J, & Goebel U. (2016). The CORM ALF-186 Mediates Anti-Apoptotic Signaling via an Activation of the p38 MAPK after Ischemia and Reperfusion Injury in Retinal Ganglion Cells. PLoS ONE, 11(10), e0165182.
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10

Quantitative Gene Expression Analysis

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Approximately 2 × 106 cells were washed, trypsinized, and processed as described above. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit; Thermo Fisher Scientific, Waltham, MA, USA). Real-time polymerase chain reactions (RT-PCR) were done with TaqMan® probe-based detection kit (TaqMan® PCR universal mastermix; Thermo Fisher Scientific). The following primers were used: HSP70 (heat shock protein 70) Hs00359163_s1, HO1 Hs01110250_m1, L-8 Hs00174103_m1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Hs02786624_g1 (all from Thermo Fisher Scientific). The PCR assays were then performed on an RT-PCR System (StepOne™; Thermo Fisher Scientific) under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 10 seconds, and 60°C for 1 minute. Reaction specificity was confirmed by running appropriate negative controls. The cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT).
Scheid S., Lejarre A., Wollborn J., Buerkle H., Goebel U, & Ulbrich F. (2022). Argon preconditioning protects neuronal cells with a Toll-like receptor-mediated effect. Neural Regeneration Research, 18(6), 1371-1377.
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