Quantitative Analysis of Adenosine Receptor mRNAs

Total cytoplasmic RNA was obtained from human lymphocytes by using the acid guanidinium thiocyanate phenol method. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay [25 (link)–28 (link)] of A₁, A_2A, A_2B, and A₃ ARs messenger RNAs (mRNAs) was performed using gene-specific fluorescently labeled TaqMan MGB Probe (minor groove binder) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR for A₁, A_2A, A_2B, and A₃ ARs was carried out with the Assays-on-Demand TM Gene expression Products NM_000674, NM_000675, NM_000676, and NM_000677 (Applied Biosystems), respectively. For the real-time RT-PCR of the reference gene, the endogenous control human β-actin was used, and the probe was fluorescently labeled with VIC™ dye (Applied Biosystems).

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Bortoluzzi A., Vincenzi F., Govoni M., Padovan M., Ravani A., Borea P.A, & Varani K. (2016). A2A adenosine receptor upregulation correlates with disease activity in patients with systemic lupus erythematosus. Arthritis Research & Therapy, 18(1), 192.

Publication 2016

TaqMan MGB Probe ABI Prism 7700 Sequence Detection System VIC™ dye

Acid Actin Assays Cytoplasmic Gene Gene expression Guanidinium thiocyanate Human Lymphocytes Mrnas Phenol Prism Real time pcr Reverse transcriptase Rt pcr

Corresponding Organization : University of Ferrara

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Messenger RNA (mRNA) levels of A1, A2A, A2B, and A3 adenosine receptors

control variables

Use of the endogenous control human β-actin for real-time RT-PCR

controls

No positive or negative controls were mentioned in the provided information

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