To measure isoform expression in tissues, several methods and platforms can be employed, each with its own advantages and specific applications. Here are the key methods based on the provided reference materials:

• Description: NanoString is a platform that uses color-coded probes to directly measure the expression of specific transcripts without the need for amplification.
• Advantages: High specificity and reproducibility, suitable for low-abundance transcripts.

Real-Time Quantitative PCR (qPCR):

• Description: qPCR is used to measure the expression levels of specific isoforms by amplifying and quantifying the target sequences.
• Advantages: High sensitivity and specificity for known isoforms, widely used for validation.

To measure the expression of isoforms in tissues, your current approach using qPCR is valid, but it requires refinement due to the lack of a reliable normalization method. Here's a more systematic approach to help you proceed:

1. Identify Isoform-Specific Splicing Patterns:

• RNA-Seq Data: Start by examining RNA-Seq data from multiple samples across different conditions or treatments. You can access public RNA-Seq data from sources like NCBI's Gene Expression Omnibus (GEO) or ENCODE to study the splicing patterns.
• Quality Control and Alignment: Perform quality control of the raw RNA-Seq data using tools like FastQC. Then, align the data to your genome of interest using tools such as STAR or HISAT2. This will allow you to map the reads to the correct locations on the genome, helping identify the exons and splice junctions.

2. Extract Alternative Splicing Isoforms:

• Use tools like Cufflinks, StringTie, or DEXSeq to quantify and assemble the isoforms. These tools will help you extract the isoforms generated due to alternative splicing in the juxtamembrane and cytoplasmic regions of your protein.
• You can use these tools to generate a list of isoforms (A1, A2, B1, B2), based on the different splice junctions present in the juxtamembrane and cytoplasmic regions.

3. Design Isoform-Specific Primers:

• Once you've identified the specific isoforms through RNA-Seq analysis, design primers for qPCR that are isoform-specific. For example, you can design primers targeting the junctions between exons for the juxtamembrane and cytoplasmic regions, ensuring they are specific to either A or B, and 1 or 2. This specificity will allow you to differentiate between the four isoforms (A1, A2, B1, B2) in your qPCR experiments.

4. Normalization Strategy:

• Housekeeping Genes: While your current setup doesn’t have housekeeping genes for normalization, you can still try to identify stable genes within your tissue samples using RNA-Seq data. Some common housekeeping genes (e.g., GAPDH, ACTB, or 18S rRNA) might be useful if they show stable expression across your conditions. Alternatively, you could use external RNA controls or synthetic spike-in controls to normalize your data.
• Relative Quantification: If no stable housekeeping genes are available, you can rely on relative quantification. For this, you would need to have a reference sample or tissue condition for comparison. This allows you to assess isoform expression relative to your control, though it’s less precise than absolute quantification.

For technical help read the paper "Article Differential and developmental stage specific abundance of Z...