PubMed · 7579671
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1995. Expression systems.. https://doi.org/10.1016/0958-1669(95)80100-6
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STALARD (selective target amplification for low-abundance RNA detection) enables isoform-level quantification of low-abundance RNAs using conventional laboratory equipment. Here, we describe steps for RNA isolation, primer design, reverse transcription, selective target amplification, and downstream analysis. The protocol couples selective pre-amplification with a quantitative reverse-transcription PCR (RT-qPCR) readout and optional nanopore sequencing. Using 1 μg input RNA and 12 pre-amplification cycles, STALARD reduces Cq values by approximately 10-12 cycles, bringing the target into a reliably quantifiable range. For complete details on the use and execution of this protocol, please refer to Jeong et al.1.
Elucidating the genetic basis of human phenotypes is a major goal of contemporary geneticists. Logically, two fundamental and contrasting approaches are available, one that begins with a phenotype and concludes with the identification of a responsible gene or genes; the other that begins with a gene and works toward identifying one or more phenotypes resulting from allelic variation of it. This paper provides a conceptual overview of phenotype-based vs. gene-based procedures with emphasis on gene-based methods. A key feature of a gene-based approach is that laboratory effort first is devoted to developing an assay for mutations in the gene under regard; the assay then is applied to the evaluation of large numbers of unrelated individuals with a variety of phenotypes that are deemed potentially resulting from alleles at the gene. No effort is directed toward chromosomally mapping the loci responsible for the phenotypes scanned. Example is made of my laboratory's successful use of a gene-based approach to identify genes causing hereditary diseases of the retina such as retinitis pigmentosa. Reductions in the cost and improvements in the speed of scanning individuals for DNA sequence anomalies may make a gene-based approach an efficient alternative to phenotype-based approaches to correlating genes with phenotypes.
Quantitative competitive reverse transcription-polymerase chain reaction has become a powerful tool for determining the amount of mRNA in cultured cells as well as in tissue. To ensure the reliability of the analysis, RNA with high purity and integrity is needed. However, when analyzing RNA samples from tumor biopsies, RNA degradation is often an inevitable problem. This causes differences in sample quality and furthermore adversely affects the quantification of gene expression. Here, we demonstrate that in partially degraded RNA samples different mRNAs are degraded to the same extent. Normalizing the expression level of a specific gene to that of a constitutively expressed gene in the same sample allows the relative quantification of this specific gene. Thus, the comparison of gene expression in RNA samples with varying integrity is possible.