Abstract
This chapter discusses about a differential display procedure with selected primers (SPR) that preferentially identifies messenger ribonucleic acid (mRNAs) of moderate to low abundance in a microscopic system. It provides good reproducibility of the multiple band patterns with independent RNA extractions, and is applicable to microscopic biological systems. Increasing the bias in favor of the identification of moderate- to low-abundance transcripts is important in studies of differential gene expression due to the high proportion of such transcripts in the total cellular mRNA pool. SPR combines the primer design (50% GC content) and reaction conditions of specific reverse transcription-polymerase chain reaction (RT-PCR) with the sensitivity of amplification product separation on a sequencing gel developed for arbitrarily primed RT-PCR differential display. SPR differs from mRNA differential display and RNA arbitrarily primed PCR (RAP-PCR) by the use of experimentally selected primer pairs that avoid the amplification of highly abundant transcripts. The chapter discusses in detail about the RNA extraction, primer sequences, and RT-PCR amplification reaction. Band selection and reamplification is also discussed. The chapter also presents an overview of cloning, sequencing, and testing for band heterogeneity. Reproducible amplification and elimination of false positives is elaborated in depth. The chapter also discusses about confirmation of differential expression.
Published Version
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