Competitive titration for probing low-abundance ion channel mRNA molecules in normal and regionally-ischaemic heart tissue.
We have measured the expression levels of a range of distinct ion channel genes in the apex/ventricle region and sino-atrial node (SAN) sub-regions of heart under conditions in which conventional Northern hybridization or ribonuclease protection methods were too insensitive or non-quantitative. The abundance of six potassium channel mRNAs was determined in relation to a single synthetic competitor RNA template which was co-reverse transcribed and PCR-amplified. By these methods we have shown that coronary artery ligation procedures which induce anoxia and ischaemic scarring in the apical region reduce amplifiable message abundance in a time-dependent, but non-specific manner. There was no evidence for any selective reduction of individual mRNA levels during this process. Despite a high reproducibility of titration endpoints, competitive RNA template amplification assays did not provide a simple marker for ischaemic damage, since it was not possible to control for tissue sample heterogeneity. We have also applied these competitive nucleic acid titration techniques to demonstrate expression of cAMP- and cGMP-gated ion channel sequences in small pieces of tissue derived from the SAN sub-region of rabbit heart. Although the ion channels encoded by these sequences are obligately coupled to intracellular signalling agonists commonly found in cardiac cells, they have not been described in functional terms within SAN or any other cardiac subregion. For rapid determination of cDNA molecular numbers, we have devised single-gene, DNA template controls to measure absolute abundance of a cAMP-gated cDNA derived from heart tissue. Competitive titration procedures therefore provide an important technique for probing gene induction and/or repression accompanying pharmacological or surgical interventions, or in progression of disease states. For rare cDNAs, they can estimate the representativeness of a given preparation prior to library construction, screening and retrieval of clones, while eliminating 'false positive' or 'false negative' signals.
Mileham
K A
KA
University of Leicester/Medical Research Council, Centre for Mechanisms of Human Toxicity, UK.
Northover
B J
BJ
Winter
A W
AW
Hundal
S P
SP
Brammar
W J
WJ
Conley
E C
EC
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D005784:Gene Amplification; D006352:Heart Ventricles / Q000737:chemistry / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D017202:Myocardial Ischemia / Q000235:genetics* / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012849:Sinoatrial Node / Q000737:chemistry / Q000378:metabolism; D017186:Titrimetry; D014158:Transcription, Genetic
D017931:DNA Primers; D007473:Ion Channels; D015221:Potassium Channels; D012333:RNA, Messenger