Multiplex pcr plus kit

Thirty genes were selected for digital RT-PCR verification of the RNA-Seq gene expression. cDNAs were synthesized from the same mRNAs processed for RNA-Seq libraries. The Qiagen multiplex PCR plus kit was used for the 1st round of amplification of 150–200 bp fragments in individual genes. For each tissue sample, four multiplex reactions were performed to cover 40 different regions in 30 genes. For the 2nd round PCR amplification, the Illumina Nextera dual-indexing primer system was used to barcode each individual sample and the PCR reaction was purified by AmpureXP and eluted in 10 μl water. Equal amount of DNA from individual libraries was pooled together for Illumina HighSeq sequencing (2 × 150 paired end).
Three sequence fragments each with an allele-specific SNP were amplified to confidently distinguish the transgenic GAI allele (Vvgai1, [14 ]) from non-transgenic one (VvGAI1, GSVIVG01011710001 [19 ]) in the ‘Thompson Seedless’ background. The ratios of Vvgai1/VvGAI1 in the three regions were then averaged and the mean ratio was used to reflect the relative qRT-PCR expression levels of the transgene Vvgai1 in a tissue. The mean ratios derived from the qRT-PCR data were further used for a correlation analysis of the RNA-Seq expression levels of individual endogenous genes with that of Vvgai1 across different transgenic vines.

The Qiagen Multiplex PCR Plus Kit was used containing the HotStarTaq Plus DNA Polymerase without any 3′-5′exonuclease activity because using a DNA Polymerase with 3′-5′exonuclease activity shows false positive results (data not shown). Since the assay comprises two multiplex reactions, the primer sequence has to be designed with similar reaction kinetics. A C/G content of 40%–60% is suggested as general guideline for specific annealing. PCR for WT and MT tubes were carried out using a thermal cycler (Eppendorf Mastercycler, vapo.protect, Germany). Final volume of PCR reaction was 25 µl. The mixture of PCR comprised 1 × Multiplex PCR Master Mix (Qiagen, Germany), 2.5 µl Primer Mix, 150 ng DNA, 0.2 µl Taq polymerase. PCR was performed within 5 min of denaturation at 95°C, 35 cycles of 95°C for 30 s, annealing at 65°C for 90 s, 72°C for 30 s, and a final extension at 68°C for 10 min.

Sixty consecutive patients referred for index case analysis or familial studies were analyzed by three methods: (i) the commercial Viennalab Realtime PCR kit based on a TaqMan assay, (ii) the improved RFLP method described here, and (iii) Sanger's dideoxy sequencing as a gold standard for accurate genotyping. All patients gave informed written consent.
The QuickGene Blood DNA extraction kit was from Qiagen (Hilden, Germany). Oligonucleotides were from Eurogentec (Seraing, Belgium). SexAI and BspHI were from New England Biolabs (Evry, France). HFE C282Y RealFast Assay and HFE H63D RealFast Assay were from ViennaLab Diagnostics (Vienna, Austria). The multiplex PCR plus kit was from Qiagen (Hilden, Germany). The MasterMix PCR AmpliTaq Gold360 was from Thermo Fisher Scientific (Waltham, MA). FastAP thermosensitive Alkaline Phosphatase was from Thermo Scientific (Vilnius, Lithuania). BigDye Terminator v1.1 cycle sequencing kit and formamide were from Applied Biosystems (Austin, TX).