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. 2014 Oct 23;19(4):601–610. doi: 10.2478/s11658-014-0214-4

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture

Raúl Loera-Valencia 1,2,✉, Xuan-Yu Wang 2, George W J Wright 2, Carlos Barajas-López 1, Jan D Huizinga 2
PMCID: PMC6275803  PMID: 25338768

Abstract

The interstitial cells of Cajal (ICC) drive the slow wave-associated contractions in the small intestine. A commonly used marker for these cells is c-Kit, but another marker named Ano1 was recently described. This study uses single-cell RT-PCR, qPCR and immunohistochemistry to determine if Ano1 could be reliably used as a molecular marker for ICC in single-cell mRNA analysis. Here, we report on the relationship between the expression of c-Kit and Ano1 in single ICC in culture. We observed that Ano1 is expressed in more than 60% of the collected cells, whereas c-Kit is found only in 22% of the cells (n = 18). When we stained ICC primary cultures for c-KIT and ANO1 protein, we found complete co-localization in all the preparations. We propose that this difference is due to the regulation of c-Kit mRNA in culture. This regulation gives rise to low levels of its transcript, while Ano1 is expressed more prominently in culture on day 4. We also propose that Ano1 is more suitable for single-cell expression analysis as a marker for cell identity than c-Kit at the mRNA level. We hope this evidence will help to validate and increase the success of future studies characterizing single ICC expression patterns.

Keywords: Interstitial cells of Cajal, c-Kit; Ano1, Multiplexed RT-PCR, Single-cell PCR, Transcriptional regulation, ICC marker, Small intestine, Primary cultures, Pacemaker cells

Full Text

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Abbreviations used

Ano1

anoctamin 1

HS

HEPES buffer saline solution

ICC

interstitial cells of Cajal

NC

no cell control

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