Table 1.
RNA extraction | cDNA synthesis | Preamplification | qRT-PCR | |
---|---|---|---|---|
Conventional workflow | ||||
Kits used | mirVana miRNA Isolation Kit | High-capacity cDNA reverse transcription kit |
TaqMan Universal PCR Master Mix |
|
Task | Extraction of total RNA | cDNA synthesis using random primers | qRT-PCR specific for the concerning targets | |
Modified workflow | ||||
Kits used | Oragene protocol + mirVana miRNA Isolation Kit | SuperScript III First-Strand Synthesis SuperMix Kit | Taqman PreAmp Master Mix Kit | TaqMan Universal PCR Master Mix |
Task | Extraction of total RNA from whole saliva (human – panbacterial) | cDNA synthesis from human RNA by using oligo(dT) primers | Unbiased and multiplex amplification of up to 100 targets | Unbiased qRT-PCR specific for the concering targets |
Rationale | Isolation of RNA from whole saliva |
poly(A) + -selected human cDNA |
Increase quantity of human cDNA | Unbiased qRT-PCR |
Also shown are the comparisons between the conventional workflow (suggested by the manufacturer) and our modified workflow, including the required kits, the tasks and the rationale for our modifications.