Gene Expression Quantification via qPCR

The mRNA levels of each gene were measured via qPCR. Following procedures previously described (50 (link)), RNA was isolated using a total RNA isolation kit, including an on-column deoxyribonuclease treatment (Norgen). cDNA synthesis was carried out using the SuperScript III Reverse Transcriptase Kit using a mixture of oligo(dT) and random hexamers for priming (Life Technologies). qPCR was conducted with Fast SYBR Green Master Mix (Applied Biosystems), and fluorescence was monitored using a 7900HT Fast real-time instrument (Applied Biosystems). Data were analyzed using the ΔΔCt method. The endogenous control transcripts were used for normalization. Statistical significance was determined using a one-tailed Student’s t test. The sequences of the primers used for all qPCR assays are in table S3.

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Yang Z., Wang W., Zhao L., Wang X., Gimple R.C., Xu L., Wang Y., Rich J.N, & Zhou S. (2021). Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs. Science Advances, 7(9), eabb0737.

Publication 2021

SuperScript III Reverse Transcriptase Kit random hexamers for priming Fast SYBR Green Master Mix 7900HT Fast real-time instrument

Assays Cdna Deoxyribonuclease Fast green Fluorescence Gene Isolation Mrna Oligo Primers Reverse transcriptase Student Sybr green Synthesis

Corresponding Organization : University of California, San Diego

Other organizations : Huzhou Women and Children's Hospital, City University of Hong Kong, West China Second University Hospital of Sichuan University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA levels of each gene

control variables

Endogenous control transcripts used for normalization
Procedures previously described (50 (link))

controls

Positive control: Not specified
Negative control: Not specified

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