High-quality total RNA isolation from cells

The total RNA was extracted from approximately 1 x 10⁷ thymocytes, peripheral CD3⁺ T cells or PILs using the mirVana total RNA isolation kit (Ambion, NY, USA) according to the manufacturer´s instructions. The parameters for the quality control of the RNA preparations were previously described [43 (link)]. RNA preparations were confirmed to be free of proteins and phenol by UV spectrophotometry.
The integrity of RNA species was assessed by microfluidic electrophoresis using Agilent 6000 RNA Nanochips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples that were free of proteins and phenol and featured an RNA Integrity Number (RIN) ≥ 9.0 were used (data not shown).

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Fornari T.A., Donate P.B., Assis A.F., Macedo C., Sakamoto-Hojo E.T., Donadi E.A, & Passos G.A. (2015). Comprehensive Survey of miRNA-mRNA Interactions Reveals That Ccr7 and Cd247 (CD3 zeta) are Posttranscriptionally Controlled in Pancreas Infiltrating T Lymphocytes of Non-Obese Diabetic (NOD) Mice. PLoS ONE, 10(11), e0142688.

Publication 2015

mirVana total RNA isolation kit Agilent 6000 RNA Nanochips Agilent 2100 Bioanalyzer

Electrophoresis Isolation Phenol Proteins Spectrophotometry T cells Thymocytes

Corresponding Organization : Clinics Hospital of Ribeirão Preto

Other organizations : SUPERA Park of Innovation and Technology of Ribeirão Preto

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Variable analysis

independent variables

Thymocytes
Peripheral CD3+ T cells
PILs (Peripheral Induced Lymphocytes)

dependent variables

Quality and integrity of RNA preparations

control variables

RNA extraction protocol using mirVana total RNA isolation kit
RNA quality control parameters as previously described [43]
Confirmation of RNA samples being free of proteins and phenol by UV spectrophotometry
RNA integrity assessment by microfluidic electrophoresis using Agilent 6000 RNA Nanochips and Agilent 2100 Bioanalyzer
Use of only RNA samples with RNA Integrity Number (RIN) ≥ 9.0

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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