Quantitative mRNA Expression Analysis

Total mRNA was isolated from A20 cells and primary B lymphocytes using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop 2000 (Thermo Scientific) and 1 µg of DNAse I treated RNA was used with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was amplified on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) using PowerUp SYBR Green Master Mix (Applied Biosystems). PCR amplification was performed by initial activation for 2 min at 50°C, followed by a 95°C 2 min melt step. The initial melt steps were then followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. Data were analyzed with the instrument software v1.3.1 (Applied Biosystems) and analysis of each target was carried out using the comparative Ct method.

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Emrich S.M., Yoast R.E., Zhang X., Fike A.J., Wang Y.H., Bricker K.N., Tao A.Y., Xin P., Walter V., Johnson M.T., Pathak T., Straub A.C., Feske S., Rahman Z.S, & Trebak M. (2023). Orai3 and Orai1 mediate CRAC channel function and metabolic reprogramming in B cells. eLife, 12, e84708.

Publication 2023

RNeasy Mini Kit NanoDrop 2000 High-Capacity cDNA Reverse Transcription Kit QuantStudio 3 Real-Time PCR System PowerUp SYBR Green Master Mix instrument software v1.3.1

B lymphocytes Cdna Cells Dnase I rna Mrna Reverse transcription Rna i Sybr green

Corresponding Organization :

Other organizations : Pennsylvania State University, New York University, University School, University of Pittsburgh

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels

control variables

RNA isolation method (RNeasy Mini Kit)
RNA quantification method (NanoDrop 2000)
CDNA synthesis method (High-Capacity cDNA Reverse Transcription Kit)
PCR amplification method (PowerUp SYBR Green Master Mix, QuantStudio 3 Real-Time PCR System)
PCR cycling conditions (2 min at 50°C, 2 min at 95°C, 40 cycles of 15 s at 95°C, 15 s at 60°C, 30 s at 72°C)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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