Quantification of XBP1 splicing

Synthesized cDNA as described above was used to measure levels of unspliced and spliced XBP1. mRNA was measured by regular PCR performed with GoTaq Green Master Mix (Promega cat. no. M7123) on a T100 Thermo Cycler (Bio-Rad, cat. no. 186-1096). The primers targeting the spliced XBP1 region (116 bp) (SI Appendix, Table S4) were designed according to a previous study (46 (link)). PCR products were resolved by agarose gel electrophoresis and visualized using GelGreen stain (Biotium, cat. no. 41005) on an ImageQuant LAS 4000 machine (GE Healthcare). Densitometric analysis of product bands was performed using the Gel Analyzer function in Fiji from NIH (47 (link)).

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Zhao G., Tan Y., Cardenas H., Vayngart D., Wang Y., Huang H., Keathley R., Wei J.J., Ferreira C.R., Orsulic S., Cheng J.X, & Matei D. (2022). Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids. Proceedings of the National Academy of Sciences of the United States of America, 119(41), e2203480119.

Publication 2022

GoTaq Green Master Mix T100 Thermo Cycler GelGreen stain ImageQuant LAS 4000 machine

Agarose gel electrophoresis Cdna Densitometric Mrna Primers Promega Stain Xbp1

Corresponding Organization :

Other organizations : Northwestern University, Boston University, Indo-American Center, Purdue University West Lafayette, University of California, Los Angeles, Los Angeles Medical Center, Jesse Brown VA Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of unspliced and spliced XBP1 mRNA

control variables

None explicitly mentioned

controls

No positive or negative controls were mentioned in the provided information.

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