Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.
A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.
Ikonomov
O C
OC
Worcester Foundation for Biomedical Research, Shrewsbury, MA, USA.
Jacob
M H
MH
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D002642:Chick Embryo; D002924:Ciliary Body / Q000294:innervation / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism*; D005724:Ganglia / Q000737:chemistry; D015723:Gene Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification*; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger