Validating internal controls for quantitative plant gene expression studies

14 Feb

Transcript level changes of a target gene, psa-h, was also evaluated by two independent normalisation strategies, by the Ribo Green method or by using multiple references.

In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary.

The polymerase chain reaction (PCR) is a common method for amplifying DNA; for RNA-based PCR the RNA sample is first reverse-transcribed to complementary DNA (c DNA) with reverse transcriptase.

The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines propose that the abbreviation q PCR be used for quantitative real-time PCR and that RT-q PCR be used for reverse transcription–q PCR.

Cells in all organisms regulate gene expression by turnover of gene transcripts (single stranded RNA): The amount of an expressed gene in a cell can be measured by the number of copies of an RNA transcript of that gene present in a sample.

In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers, deoxyribonucleotides, a suitable buffer solution and a thermo-stable DNA polymerase.

A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at the required wavelength allowing the generation rate to be measured for one or more specific products.

A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (q PCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).