ML243

Reverse transcription technology uses RNA as a template to synthesize DNA. RT-PCR is simple, specific and sensitive, and can be used to detect gene expression levels and expression differences in cells; detect RNA virus content; clone cDNA sequences of specific genes.

Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Northern blot detects a defined RNA species by separating denatured RNA by size, transferring RNA to a membrane, hybridizing with a complementary labeled DNA or RNA probe, and detecting probe-bound RNA by autoradiography, phosphorimaging, or validated nonradioactive detection. The readout is both RNA size and abundance: band migration estimates transcript length or RNA-processing state, while band intensity reflects relative target RNA amount after normalization to total RNA, rRNA, or another validated loading control. In cancer cells, primary neurons, mouse tumor samples, intestinal organoids, inflammatory macrophages, or drug-screening studies, Northern blot is most appropriate when transcript size, isoform pattern, RNA processing, or small-RNA detection is important; qPCR or RNA-seq can complement it when higher sensitivity or global profiling is needed.

Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A, luminal B, HER2-positive and basal-like. Molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer.

Real-time quantitative PCR (qPCR) quantifies an amplifiable nucleic-acid target by monitoring fluorescence during PCR cycling rather than measuring product only after amplification. The increase in fluorescence tracks accumulation of PCR product, and the quantification cycle (Cq; historically also Ct/CP) is related to the initial amount of target: samples containing more starting target generally reach the defined fluorescence threshold in fewer cycles.