Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) and following the manufacturer’s instructions. Then, cDNA was synthesized from total RNA (1 μg) and following the given instructions (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA). For real-time PCR, cDNA (50 ng) was amplified with Inventoried TaqMan Gene Expression Assay products (MIP-1α: Hs04194942_s1; GAPDH: Hs03929097_g1; TLR4: Hs00152939_mL; and IRF3: Hs01547283_m1) comprising two gene-specific primers, one TaqMan MGB probe (6-FAM dye-labeled), and TaqMan® gene expression master mix (Applied Biosystems) using a 7500 fast real-time PCR system (Applied Biosystems). MIP-1α mRNA expression was normalized against GAPDH mRNA expression and target gene expression relative to control was calculated by using 2−ΔΔCT method, expressed as fold change over average gene expression in control treatment taken as 1.
Taqman mgb probe 6 fam dye labeled
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan MGB probe (6-FAM dye-labeled) is a molecular biology tool designed for use in real-time PCR (polymerase chain reaction) assays. It is a sequence-specific oligonucleotide probe that contains a fluorescent reporter dye (6-FAM) and a minor groove binder (MGB) moiety. The MGB moiety increases the melting temperature of the probe, allowing for the use of shorter probe sequences while maintaining specificity.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Inventoried taqman gene expression assay products, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
3 protocols using taqman mgb probe 6 fam dye labeled
1
qRT-PCR Analysis of Immune Markers
2
Quantifying Target Gene Expression via qRT-PCR
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Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions [44 (link)]. Complementary DNA (cDNA) was synthesized using 1 μg of total RNA following the guidelines from the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). For each real-time PCR reaction, 50 ng of cDNA template was amplified using Inventoried TaqMan Gene Expression Assay products (MCP-1: Hs00234140_m1; ACSL-1: Hs00960572_g1, GAPDH: 4310884E) using two gene-specific primers, one TaqMan MGB probe (6-FAM dye-labeled), TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA), and a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) [28 (link)]. The target mRNA levels were normalized against GAPDH mRNA relative to the control and calculated using the 2−ΔΔCT method [45 (link),46 (link),47 (link),48 (link)]. Relative mRNA expression was expressed as fold expression relative to the average of control gene expression. The expression level in the controls was designated as 1 [49 (link),50 (link)].
3
Quantitative Analysis of Cytokine Expression
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Total RNA was extracted from treated and untreated THP1 cells using RNeasy Mini Kit (Qiagen, Valencia. CA, USA). One microgram of total RNA was used for cDNA conversion using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). Real time polymerase chain reaction was performed using 50ng of cDNA, gene specific TaqMan Gene Expression Assays containing gene-specific primers and TaqMan MGB probe (6-FAM dye-labeled) and TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster city, CA, USA) in a QuantStudio 5Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The Ct values of CCL4 and SAPK/JNK were normalized with corresponding GAPDH Ct values and the expression level of CCL4 in treated samples relative to control samples were calculated with -2ΔΔCt-method. Relative mRNA expression was expressed as fold expression over average of control gene expression. The expression level in control treatment was assumed to be 1.
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