A sample was only considered positive if both the sample quantification curve became exponential, and the PCR product was correctly differentiated by the Tm analysis step.
Total RNA was used to amplify HIF-1α/VEGF/β-actin using the Superscript TM One Step RT-PCR with Platinum®Taq (Invitrogen, Sydney, Australia).
RNA was extracted from serum using silica-membrane-based nucleic acid purification kit (QIAmp viral RNA, Qiagen, Germany), RNA was reverse transcribed (Superscript TM One step RT PCR Platinum Taq; Invitrogen, Germany) and specific primers for nucleotide (nt) 883 906 and 1288 1265 positions of HDV genome were used [ 30].
Detection of viral nucleic acids using real-time PCR (qPCR) and real-time rt-PCR (rt qPCR) CPV DNA was amplified by qPCR using TaqMan® Gene Expression 2× Master Mix Applied Biosystems CDVCDV and CCoV RNA were amplified by rt-qPCR using the TaqMan® RNA-to-Ct(TM) 1 step kit in a 20 μl reaction with 50 ng of template.
(This substantial difference is expected to be even larger in a setting where an expressed Cre does not require a second, TM-mediated step for activation, as is the case with TM-independent Cre expressed from the Prm promoter, but not with TM-inducible CreER, expressed from the Prp promoter).
Immunohistochemical staining was performed using the Superision TM two-step method.
The latter stands for the worst case condition, in that, for a given pair {r,m}, the search is halted as soon as a feasible TM is found (step 10 of Table 2), because any other TM with the same pair {r,m} would require a larger amount of transmitted power.
Immunohistochemical staining was performed to detect the NET-1 and Ki67 expressions in the sections using Dako Elivision TM Plus Two-step System PV-60000 kit, Zymed, Co. USA).
Dysfunction of FOXC1 contributes to the death of TM cells, an important step in the development of glaucoma.
The PCR cycles were finished with an annealing step at Tm pp-5 for 1 minute and an extension step at 72°C for 30 minutes.
