Reverse transcription-polymerase chain reaction (RT-PCR)
Objectives
- To detect and quantify mRNA expression using a most sensitive technique
- Can be used to quantify mRNA levels from smaller samples
- Allows the determination of mRNA expression in various tissues
- Allows the comparison of mRNA expression between normal and pathophysiological, treated or untreated conditions
Summarized methodology
Several steps are to be taken into account in order to optimize the results to be obtained:
- Tissue harvesting and homogenization in RNase free conditions
- Extraction of total RNA from tissue using the TRIzol reagent method (checking of the absence of genomic DNA contamination)
- Amplification of cDNA using specific primers
- Loading of PCR product onto an agarose gel for electrophoresis
Endpoints
Measurement of changes in gene expression between different physiological or pathophysiological, treated or untreated conditions using relative quantification i.e. comparing mRNA expression levels between multiple samples, using an internal control (gene coding for ubiquitous protein expression i.e Beta 2-microglobulin or GAPDH) for sample normalization.
NB: Pelvipham will gladly study the feasibility of evaluating the expression and activity of other proteins to meet its client’s needs.
Links to applicable Targeted disorders / Pathophysiological models
- Atherosclerosis
- BPH (Benign Prostatic Hyperplasia)
- Diabetes
- ED (Erectile Dysfunction)
- Ejaculatory disorders (premature or delayed ejaculation / anejaculation)
- FSD (Female Sexual Dysfunction)
- Hypertension
- IC (Interstitial Cystitis) / Painful bladder syndrome
- Metabolic Syndrome and Obesity
- Myocardial Infarction
- NDO (Neurogenic Detrusor Overactivity)
- OAB (Overactive Bladder)
- SCI (Spinal Cord Injury)
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