Mesa green qpcr mastermix plus for sybr assay low rox

Two real-time PCR techniques were used to detect a possible deletion in IDUA and GAA (http://www.eurogentec.com/8233Discover+our+new+qPCR+guide.html). First, TaqMan Probe assays were designed using Primer Express 2.0 (Applied Biosystems) and performed on an ABI 7500, using qPCR low ROX MasterMix Plus (Eurogentec). For IDUA exons 1–14 were tested and for the GAA gene exon 3, 8, 13 and 18. Subsequently, results were verified with SYBR green assays on an ABI 7900 using MESA GREEN qPCR MasterMix Plus for SYBR assay Low Rox (Eurogentec) for exon 2 of the IDUA gene and exon 13 of the GAA gene using the TaqMan assay outer primers.

Real-time (reverse transcriptase) PCR for mRNA was done with the Mesa Green qPCR Mastermix Plus for SYBR Assay-Low ROX (Eurogentec) by using the 100–200 ng of the total RNA with the primers listed in Supplementary Table 2. The endogenous control used was 18S ribosomal RNA. For the quantification of miRNA, 30 ng of total RNA was taken. Following primers were used for the amplifications of different miRNAs. For example, human let-7a (assay ID 000377), human miR-122 (assay ID 000445), human miR-122* (assay ID 002130), human miR-16 (assay ID 000391), human miR-21 (assay ID 000397), human miR-24 (assay ID 000402), human miR-125b (assay ID 000449), U6 snRNA (assay ID 001973) and Applied Biosystem Taqman chemistry-based miRNA assay system were used for the experiment. All reactions were done in 7500 Applied Biosystem Real Time System or BIO-RAD CFX96 Real-Time system. Cycles were set as per the manufacturer's protocol.