Isolation of mRNA Species Enriched in the Ocular Tissues of Form-Deprived Chick Eyes Using Differential Display

Abstract

We conducted studies with mRNA differential display technology using mRNA from control and form-deprived ocular tissues of chicks. That is, each mRNA was reverse-transcribed with oligo-dT primers anchored to the beginning of the poly(A) tail and amplified by polymerase chain reaction using 10-mer oligonucleotides arbitrary in sequence as second primers. When amplified products were electrophoresed on a denaturing polyacrylamide gel, approximately 10–15 bands enriched in form-deprived ocular tissues were obtained for each second primer. The products eluted from the gel were subjected to secondary amplification and used as probes in the Northern blot analysis. When Northern blot analysis confirmed that the intense bands reflected mRNA species enriched in form-deprived ocular tissues, the products were force-cloned into pGem T vector (Promega) and subjected to sequence analysis. Until now, 21 different clones were isolated, although their sequences did not always display high homology to previously reported sequences in the DNA databases. It is not yet known whether form-deprivation induces mRNA transcription or decreases mRNA turnover. However, alteration in mRNAs by form deprivation might include signals related to the progression of experimental myopia.