Methods to measure gene expression in mammalian cells in vitro and in vivo - Essay Example

This method is based on suppression PCR technique that is a combination of normalization and subtraction in one procedure. The normalisation technique would equalize cDNA abundance in the target population while the subtraction step excludes the sequences that are common between the target and driver populations. 5 The two type of real – time PCR are the molecular beacon and SYBR ® Green method. SYBR ® Green method is the first method that was used in real – time PCR wherein it binds to double – stranded and when this would get excited, would emit light.

On the other hand, molecular beacon utilizes a reporter probe that is wrapped to the hairpin.9 Involves mRNA isolation and hybridisation. mRNA is extracted and purified from the cells. To proceed with electrophoresis, mRNA is loaded to the gel. The current is allowed to pass through the gel and mRNA will move away from the negative electrode. To visualize mRNA, Flourescent dye is used as a stain followed by UV lighting. RNA is then transferred to a membrane from the gel electrically or through capillary action using a high – salt solution.

RNA fragment that is in question is incubated with the blot and to remove the probe, the blot is then washed. Developmental step follows.10 A method to analyse comprehensively patterns of gene expression.8 mRNA is isolated from the sample. A small chunk of sequence is then extracted and these small pieces are sequenced together forming a long chain. These chains are cloned into a vector that can be taken up by bacteria. These chains are sequenced using a modern high. The data is then processed with computer in order to count small tags sequence.

13 This method works at the 3 terminal portions of mRNAs by systemic amplification and resolution on DNA sequencing gel fragments. Primers are anchored and are designed to bind to 5 boundary of the poly-A tails for the reverse transcription.