Commonly used terms in PCR
To ensure reliable results in real-time PCR, it is essential that the WTA method provides unbiased and accurate amplification of the whole transcriptome. This means that the sequence and the relative abundance of each transcript should be preserved after WTA, otherwise false results in gene expression analysis will occur. The principle of WTA is shown in the figure Schematic representation of whole transcriptome amplification.
As reverse transcription takes place using a mix of random and oligo-dT primers, a cDNA library covering all transcript sequences, including both 5' and 3' regions, is prepared. Subsequent ligation of the cDNA followed by MDA using a uniquely processive DNA polymerase generates amplified cDNA that preserves the transcript representation of the original RNA sample. This is critical for accurate gene expression analysis.
When carrying out WTA, it is important to consider both the amount of starting material (i.e., the number of cells or the amount of RNA) and the copy number of the transcripts of interest. The table Transcript representation in different cell amounts shows the relationship between the amount of starting material and transcript representation (note that this is only a guide: the number of transcripts per given amount of starting material can vary). In starting material where the copy number of a transcript is 10 or less (highlighted in bold in the table, stochastic problems will occur (i.e., the unequal distribution of a very low number of transcripts in a highly dilute solution). This may result in underrepresentation of the low-copy transcript at the start of WTA. Special consideration should be given to mosaic transcripts, which are derived from genes that are expressed only in a subset of cells in tissues. Since these transcripts are not present in every cell, they will not be accurately represented in low amounts of starting material (i.e., 1–102 cells).
Reliable WTA depends on the copy number of the transcripts. 10 ng of RNA corresponds to about 500 cells, and even low-copy transcripts are well represented in this amount of RNA. Using lower amounts of RNA or a very limited number of cells means that the starting material could have a partial representation or an absence of low-copy transcripts.