Isoform-specific Primers
Isoform-specific Primers - Related Videos
11:19
Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets
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Cited by 13 •
2010
Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.
10:17
Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers
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Cited by 7 •
2017
Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.
01:22
Analysis of Glutamic Acid Decarboxylase Isoforms in Human Brain Tissue
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2025
Homogenize the tissue with a homogenizer to release proteins, including glutamic acid decarboxylase, or GAD enzyme isoforms, essential for brain function.
11:14
Primer-Free Aptamer Selection Using A Random DNA Library
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Cited by 20 •
2010
SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.
07:08
A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
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Cited by 3 •
2016
We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.
FAQs
Selectivity comes from placing one primer within a sequence that distinguishes the desired transcript from other isoforms. A primer can target a unique exon or another isoform-specific region, allowing amplification only when the complementary sequence is present. This design connects the PCR signal to one transcript variant rather than to the gene as a whole.
An exon–exon junction can provide a sequence arrangement associated with a particular processed transcript. Positioning a primer across a distinguishing junction helps favor amplification of that isoform during reverse-transcription PCR or quantitative PCR. The resulting assay is better suited to separating alternatively spliced transcripts whose exon combinations differ.
A transcript-level signal can show that individual isoforms change differently even when they originate from the same gene. This distinction supports analysis of alternative splicing and isoform-specific expression across tissues, developmental stages, or disease states. It can therefore expose regulatory or biological differences that a combined gene-level measurement would obscure.
The workflow begins by selecting a sequence feature unique to the transcript of interest, such as a distinguishing exon or exon–exon junction. RNA is then examined through reverse-transcription PCR or quantitative PCR using the designed oligonucleotides. Amplification or quantitative expression results are interpreted as evidence for the presence or relative expression of that isoform.
Isoform-specific primers provide a targeted experimental check for transcript variants suggested by RNA sequencing. By testing amplification of the relevant isoform in reverse-transcription PCR or quantitative PCR, researchers can examine whether the predicted transcript is present and compare its expression across samples. This adds focused validation to sequence-based transcript analysis.
These assays support comparisons among tissues, developmental stages, and disease states, where alternative isoforms may show different expression patterns. The measurements can help investigate gene regulation and possible functional differences among transcript variants. In applied biology, isoform-specific expression patterns may also point to candidate biomarkers or therapeutic targets.