Reverse transcription 2 system

Manufactured by Promega

Sourced in United States

The Reverse Transcription II system is a laboratory tool used to convert RNA into complementary DNA (cDNA) for various downstream applications. The system provides the necessary reagents and protocols to perform this reverse transcription process.

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Lab products found in correlation

Abi prism sequence detection system, by Thermo Fisher Scientific (3 mentions) Rneasy kit, by Qiagen (3 mentions) Vic labeled probe, by Thermo Fisher Scientific (1 mentions) Oligo dt 15, by Promega (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Abi 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions) Abi sequence detection system, by Thermo Fisher Scientific (1 mentions)

4 protocols using reverse transcription 2 system

Quantitative RT-PCR Analysis of C3 and GAPDH

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Quantitative RT-PCR analysis was performed to determine the mRNA expression levels of C3 and GAPDH. The total RNA was harvested from the serum of all individuals using an RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Briefly, the PCR procedures were as follows: Pre-denaturation at 95°C for 30 sec, followed by 35 cycles of denaturation at 95°C for 15 sec and annealing at 56°-60°C for 30 sec. RT-PCR experiments were performed a minimum of three times.
RNA (1 μl) was reverse transcribed into cDNA using random primers in a Reverse Transcription II system (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. The mRNA expression levels of C3 and GAPDH were determined using quantitative PCR with an ABI Prism Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers used are listed in Table I. An assay reagent containing premixed primers and a VIC-labeled probe (Applied Biosystems Life Technologies; cat no. 4310884E) was used to quantify the mRNA expression level of endogenous GAPDH. The relative levels of C3 transcripts were normalized against the amount of GAPDH mRNA for each sample.

JIANG H., GUO M., DONG L., CAO C., WANG D., LIANG X., GUO F., XING Z., BU P, & LIU J. (2014). Levels of acylation stimulating protein and the complement component 3 precursor are associated with the occurrence and development of coronary heart disease. Experimental and Therapeutic Medicine, 8(6), 1861-1866.

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Quantitative Analysis of Ad36-Induced Adipogenic Genes

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The total RNA was isolated from Ad36 infected preadipocytes using Trizol (Invitrogen Corporation, USA). The RT-PCR experiments were repeated at least 3 times. RNA (1 μg) was reverse-transcribed into cDNA using Oligo (dT)15 in a reverse transcription II system (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Expression of mRNAs was quantified by quantitative PCR using an ABI Prism Sequence Detection System (Applied Biosystems). Primers for β-actin, APMI, Visfatin and E4orf1 genes were given in Table 1. Template-negative and RT-negative conditions were used as controls. Gene amplification was monitored using the ABI 7500 software. The corresponding amplification plots were used to determine the threshold cycle value. And the threshold cycle value was defined as the number of PCR cycles taken for fluorescent intensity to reach a fixed threshold for each signal. The relative amounts of APMI, Visfatin and E4orf1 mRNA were normalized to the amount of β-actin mRNA in the same sample.

Jiao Y., Mao X., Chang X., Abudureyimu K., Zhang C., Lu J., Wang Y., Nuermaimaiti N., Aisa Y., Gong X, & Guan Y. (2014). Adenovirus36 infection expresses cellular APMI and Visfatin genes in overweight Uygur individuals. Diagnostic Pathology, 9, 83.

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Quantification of MMP-2 and MMP-9 Expression

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Mice were euthanized by CO₂ inhalation following completion of the experiments and the spinal cords were quickly dissected for RNA isolation using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. One microliter of RNA was reverse transcribed into cDNA using random primers with a Reverse Transcription II system (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. PCR was conducted using an ABI Prism Sequence Detection System (Applied Biosystems, Foster City, CA, USA). A VIC^®-labeled probe (cat. no. 4310884E; Applied Biosystems) was used to quantify the expression of endogenous GAPDH mRNA, which was used as an internal control. Amplification of the MMP-2 and MMP-9 cDNAs and the endogenous GAPDH cDNA were determined using FAM™ and VIC fluorescence, respectively. The relative amounts of MMP-2 and MMP-9 transcripts were expressed as ratios relative to the levels of GAPDH mRNA. The experiments were repeated independently at least three times. The primers used for MMP-2 were 5′-GGAGCA CGTCATGCAC and 5′-AGACACGCTAGTAGGC, and for MMP-9 were 5′-CACCACTGCAATTGCG and 5′-CACCAT CTCATACGT GAG.

ZHANG H., CHU G., PAN C., HU J., GUO C., LIU J., WANG Y, & WU J. (2014). A nutrient mixture reduces the expression of matrix metalloproteinases in an animal model of spinal cord injury by modulating matrix metalloproteinase-2 and matrix metalloproteinase-9 promoter activities. Experimental and Therapeutic Medicine, 8(6), 1835-1840.

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Quantifying Immune Gene Expression in PBMCs

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Total RNAs were harvested from the peripheral blood mononuclear cell samples using the RNeasy kit (Qiagen Inc., Valencia, CA, USA), according to the manufacturer’s instructions. The RT-qPCR experiments were repeated for a minimum of four times. Subsequently, the total RNA samples (1 μl) were reversely transcribed into cDNA using random primers in a Reverse Transcription II system (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Next, the mRNA expression levels were determined by qPCR using an ABI Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). An assay reagent containing premixed primers and a VIC-labeled probe was used to determine the mRNA expression levels of endogenous GAPDH. The FAM fluorescent intensity was measured to detect the cDNA expression levels of IL-2 and IL-10, while the VIC fluorescent intensity was measured to determine the cDNA expression of endogenous GAPDH, using the ABI 7900HT Fast Real-Time PCR system (Applied Biosystems Life Technologies). The relative mRNA expression levels of IL-2 and IL-10 were normalized to the level of GAPDH mRNA of each individual. The primers used in RT-qPCR are listed in Table II.

LIANG C., DU W., DONG Q., LIU X., LI W., WANG Y, & GAO G. (2015). Expression levels and genetic polymorphisms of interleukin-2 and interleukin-10 as biomarkers of Graves’ disease. Experimental and Therapeutic Medicine, 9(3), 925-930.

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