A master mixture of QNASBA (Quantitative Nucleic Acid Sequence Based Amplification) was used to quantify marker proteins by real time amplification of the aptamers.
The cDNA was amplified using a SYBR Green master mixture (Bio-Red, USA) with a LightCycler 96 (Roche, USA).
Care was taken to avoid contamination by doing all procedures like isolation of DNA, PCR master mixture preparation, addition of template, PCR, and gel electrophoresis in separate rooms.
The total volume of the PCR master mixture was 20 μl, to which cDNA template equivalent to 25 ng RNA starting material and 0.5 μM of each primer (Table 1) was added.
The reaction mixtures were prepared using LightCycler Fast Start DNA master mixture for SYBR Green I, 0.5 µM of each primer, 4 mM MgCl2.
QPCR reactions consisted of 6 µL of 454 library as DNA template, 1.5 µL of primers (500 nM final conc)., and 7.5 µL of Quantitect SYBR Green master mixture (Qiagen).
FastStart DNA Master Hybridization Probes kit (Roche Molecular Biochemicals, Mannheim, Germany) was used; 4 µl of the master mixture and 5 µl of DNA samples were loaded into glass capillary tubes.
Real-time PCR reactions contained 1 µl homogenized nymphal suspensions, 1 µM 5' flaB primer, 1 µM 3' flaB primer, 0.1 µM flaB probe, and 1X TaqMan universal PCR master mixture in a total volume of 25 µl.
Real-time PCR reactions, performed in triplicate, contained 1 µl cDNA, 1 µM 5' primer, 1 µM 3' primer, 0.1 µM probe, and 1X TaqMan universal PCR master mixture in a total volume of 12.5 µl.
A 20 µL of 1× SYBR master mixture (ABI, Foster City, CA) contains 5 µM of forward/reverse primers and 20 ng of genomic DNA obtained either before or after immunocapture-amplified DNA as described above.
Real time PCRs were performed in triplicates in a total volume of 25 µl using brilliant SYBR green PCR master mixture (Stratagene) on a 7300 Real-Time-PCR System (Applied Biosystems) in 96-well PCR plates (Applied Biosystems).
