One-step reverse transcription polymerase chain reaction for semiquantitative analysis of mRNA expression.
Analysis of mRNA expression is one of the main targets of scientific research. However, its quantification can be difficult, especially when dealing with low-expression mRNAs (ionic channels, carriers, receptors, etc.) or when only small samples are available (human biopsies). Here we suggest an easy, rapid and reliable method to assess semiquantitative changes in mRNA that combines several technical improvements: i) one-step reverse transcription polymerase chain reaction (RT-PCR) from total RNA; ii) addition of ethidium bromide to the gel, which provides a more homogeneous binding to DNA; iii) direct capture of the gel image using a charged-coupled device camera and then saving the image on computer before quantification, which increases resolution and thus improves and shortens the analysis; and iv) the use of 18S rRNA as a control, which is especially useful when samples from activation, differentiation and proliferation models are used. The technique was validated by checking the system conditions of image capturing and quantification. This was corroborated by a study of Kvl.3 ion channel expression in the brain. In these conditions, the wide range of PCR cycles and total RNA allows us to correlate relative gene expression and direct input of the target gene.
Fuster
G
G
Membrane Protein Molecular Physiology Group, Department of Biochemistry and Molecular Biology, Universitat de Barcelona, Barcelona, Spain.
Vicente
R
R
Coma
M
M
Grande
M
M
Felipe
A
A
D000818:Animals; D001921:Brain / Q000378:metabolism*; D051379:Mice; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger