l10 Northern Blotting Techniques in Genetics

Northern Blotting

• RNA immobilized on a membrane is incubated with a radioactive or fluorescent probe complementary to the gene of interest.
• Visualization of the probe indicates the presence and size of RNA using a size marker or ladder.
• A Northern blot can confirm specific gene expression patterns, as exemplified by Riordan et al.'s analysis of CFTR in 1989.
• Gel electrophoresis of total RNA, termed 'Northern,’ transfers RNA from gel to a nitrocellulose membrane, facilitating specific transcript detection.
• Northern blotting demonstrates relative quantities and sizes of specific RNA transcripts but is time-consuming and requires substantial RNA input.

Fluorescent Dyes and Probes

• SYBR Green I is a cyanine dye that intercalates with double-stranded DNA, increasing fluorescence up to 1,000-fold upon binding.
• Taqman probes are dual-labeled with a dye and quencher, designed for specific hybridization to PCR products.
• During PCR cycles, the Taq polymerase cleaves the probe, releasing the dye and allowing fluorescence which increases with PCR product accumulation.

Quantitative Real-Time PCR (QPCR)

• QPCR generates real-time amplification plots, with the point where the plot crosses a threshold known as the Ct value (cycle threshold).
• A standard curve enables estimation of unknown sample quantities based on their Ct values.
• Ct values from serial dilutions (e.g., 10^8 to 10^1 copies) are plotted to create a standard curve for quantification.

Advantages and Disadvantages of QPCR

• An advantage of absolute QPCR is the elimination of the need to co-amplify housekeeping genes for every sample.
• A disadvantage includes requiring previously quantified cDNA for setting up standard curves.

Subtractive Library Screening

• This method detects differentially expressed genes between two tissues or states by mixing cDNAs from target and control tissues.
• It involves adding adapters to cDNAs of interest and subtracting similar genes to isolate unique genes.
• Subtractive screening serves as an early technique for analyzing differential gene expression (DGE).

cDNA Libraries

• cDNA libraries consist of cDNAs obtained from mRNA of a specific cell, tissue, or organism, providing a stable representation of the transcriptome during sampling.
• These libraries can be amplified via bacterial cloning with plasmid vectors, and are utilized in differential gene expression analysis.
• The cloned cDNA library typically reflects only the most highly expressed genes.

Large-Scale Genomics for Gene Expression

• Two primary technologies for measuring gene expression include gene expression microarrays and RNA sequencing.
• Gene expression microarrays feature thousands of DNA probes corresponding to all known genes, fixed to a solid surface for hybridization with cDNA, generating fluorescent signals.
• RNA sequencing involves isolating RNAs, preparing libraries by fragmenting and generating cDNA, and performing high-throughput sequencing for analysis.

This quiz covers the principles and techniques of Northern blotting, focusing on the visualization of RNA through probes. Learn how to interpret results, including RNA size and presence as shown through various imaging methods, and the importance of size markers. Perfect for students in genetics courses.