The primers of the TaqMan PCR used were: Forward primer: 5'-TTTGGTCAAGTCCTGCAACGA-3', Reverse Primer: 5'-CCCCACCTTCCTCCCAGTTA-3' and probe: FAM-TTACTAACATTAAGTTGAGGACTCTA-MGB, targeting the M. hominis 16 rRNA gene sequence [ 8], and further confirmed by specific culture (Mycoplasma IST, bioMérieux, France).
Primers and a minor groove binder (MGB) TaqMan probe were targeted to the four genes and to an unamplified reference gene selected from each of the two intervals (CNTNAP1 and UNC5ND, respectively).
For this, new set of primers and corresponding TaqMan® Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1).
Real-time RT-PCR was performed using Applied Biosystems assays-on-demand 20× assay mix of primers and Taq-Man MGB probes (FAM™ dye-labeled) for target gene TRAIL (ID Hs00234355_m1) and predeveloped 18S rRNA VIC™ dye-labeled probe) TaqMan® assay reagent (P/N 4319413E) for endogenous control.
We used Applied Biosystems (Foster City, CA) assay-by-design and assay-on-demand 20× assay mixes of primers and TaqMan MGB probes (FAM™ dye-labeled) for our target genes (IL-10) and a pre-developed 18S rRNA (VIC™-dye labled probe) TaqMan® assay reagent for an endogenous control.
Commercially available TaqMan primers and probes, including 2 unlabeled PCR primers and 1 FAMTM dye-labeled TaqMan MGB probe, were used for all the targets (all from Applied Biosystems).
The reaction from each pool was then quantitated using a NanoDrop spectrophotometer and 100 ng of the cDNA reaction products were added to 18 individual target-specific TaqMan Gene Expression Assays (TaqMan MGB probes, FAM dye-labeled according to the manufacturer's protocol) using GAPDH and ACTB as endogenous references and TaqMan Universal PCR Master Mix (Roche Molecular Systems, NJ, USA).
The CCL3L1 MGB probe sequence is as follows MGB-6FA: TTCGAGGCCCAGCGACCTCA.
TaqMan-MGB allelic discrimination assays targeting these SNPs were designed and experimentally screened against synthetic RNA templates and genomic RNAs.
In fact, a DNA probe with conjugated MGB groups forms a stable duplex with a single-stranded DNA target; therefore it can be shorter than those without MGB groups.
