A simple RT-PCR-based strategy for screening connexin identity

Abstract

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from multi primers (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the multi primer method to several human connexins.

Localization of connexin 30 in the luminal membrane of cells in the distal nephron

“…Two microliters of cDNA were amplified using a master mix containing Taq polymerase (Invitrogen) and the following primers: Cx30 sense, 5Ј-GGCTTGGTTTTCAGAGATAG-3Ј; Cx30 antisense, 5Ј-GAGTTGTGTTACCTGCTGC-3Ј; ␤-actin sense, 5Ј-GGT-GTGATGGTGGGAATGGGTC-3Ј; and ␤-actin antisense 5Ј-ATG-GCGTGAGGGAGAGCATAGC-3Ј (each at a final concentration of 200 M). Cx30 oligonucleotides were based on previously published primer sequences (30). Previous work using the same Cx30 primer sequences demonstrated the presence of Cx30 mRNA in the mouse brain (6).…”

“…The ␤-actin oligonucleotide sequence was determined using a published sequence and confirmed with sequencing. The PCR reaction was carried out for 30 cycles of the following: 94°C for 30 s, 55.4°C for 30 s, and 72°C for 30 s. The PCR product was analyzed on a 2% agarose gel stained with ethidium bromide to identify fragments of ϳ369 bp for Cx30 (30) and 400 bp for ␤-actin.…”

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

“…Total RNA from the samples was extracted using TRIzol reagent (GIBCO-BRL) and was quantified as previously described (52). Total RNA (10 g) from the samples was separated on 1.2% formaldehyde-agarose gel and was transferred onto a Gene Screen hybridization transfer membrane (NEN Life Science Products, Boston, MA).…”

“…RT-PCR was performed as previously described (52). For semiquantitative PCR, a 9:1 ratio of competimers and 18S primers (Ambion, Austin, TX) was added to the PCR mixture.…”

Blockade of Gap Junctions In Vivo Provides Neuroprotection After Perinatal Global Ischemia

“…formed on total RNA isolated from hippocampal slice cultures 17 with the Thermoscript RT-PCR system (Gibco BRL). Reaction products were analyzed by electrophoresis on 2% agarose gels.…”