Two-Step versus One-Step RNA-to-CT 2-Step and One-Step RNA-to-CT 1-Step: validity, sensitivity, and efficiency.
Quantitative RT-PCR can be carried out as a one- or a two-step reaction. However, the choice of method raises controversy from the perspective of the researcher and manufacturer, because of advantages and disadvantages with both systems. We therefore hypothesize that running the RNA-to-C(T) 2-Step kit [(Applied Biosystems (AB), Foster City, CA] using a one-step protocol (as recommended) is not appropriate for quantitation of gene expression levels and should not be performed. Consequently, we ran comparative studies of the two suggested methods to evaluate their efficiency, sensitivity, and accuracy. To ensure precession, two different PCR machines were used: the StepOnePlus system and Chromo4. In addition, the RNA-to-C(T) 1-Step kit (recently launched by AB) was also used to compare its efficiency with these methods. Efficiency, sensitivity, and linearity were determined by standard curves generated using RNA isolated from C2 myoblasts to amplify the housekeeping gene GAPDH. When the RNA-to-C(T) 2-Step kit was run as a two-step reaction on the Chromo4 or StepOnePlus, respectively, not only did the efficiency increase (100+/-1.5% and 99.7+/-0.95%) but also the sensitivity (comparative threshold cycle for the lowest standard: 33.2+/-0.5 and 32.5+/-0.7) and linearity (0.997+/-0.001 and 0.993+/-0.006) compared with RNA-to-C(T) 2-Step run as one-step and RNA-to-C(T) 1-Step kit. This is the first study to demonstrate that the RNA-to-C(T) 2-Step kit is not reliable to be performed as a one-step reaction but as a two-step reaction, is even more sensitive than the newly launched RNA-to-C(T) 1-Step kit.
Al-Shanti
Nasser
N
Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester, M1 5GD, United Kingdom. n.al-shanti@mmu.ac.uk
Saini
Amarjit
A
Stewart
Claire E
CE
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D051379:Mice; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases