Taqman universal pcr master mix reagent

Total liver RNA was extracted using the ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA). The levels of messenger RNAs (mRNAs) encoding Bsep, multidrug resistance-associated protein 2 (Mrp2), Na^+/taurocholate cotransporter (Ntcp), Fxr, cholesterol 7 alpha-hydroxylase (Cyp7a1), 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr), low-density lipoprotein receptor (Ldlr), ATP-binding cassette sub-family G member 5 (Abcg5), sterol regulatory element binding transcription factor 2 (Srebf2), collagen type I, alpha 1 (Col1a1), collagen type I, alpha 2 (Col1a2), tissue inhibitor of metalloproteinase 1 (Timp1), matrix metallopeptidase 3 (Mmp3), transforming growth factor, beta 1 (Tgfb1), intestinal bile acid-binding protein (I-babp), transmembrane G protein-coupled receptor 5 (Tgr5), Niemann–Pick C1 like 1 (Npc1l1), and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in liver tissue were determined by fluorescence-based reverse transcriptase-polymerase chain reaction (RT-PCR) using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the TaqMan Universal PCR Master Mix reagent (Applied Biosystems). The primers and probes (Applied Biosystems) are listed in S1 Table. The mRNA levels were normalized to the level of the endogenous control, Gapdh, using a primer and probe for GAPDH (4326317E, Applied Biosystems).

As the target panel is not designed to detect Schistosoma spp., which is a frequent helminth in migrant patients, we further evaluated whether the DNA extracts obtained with the MICROLAB^® STARlet device were suitable for Schistosoma detection with our in-house S. mansoni PCR method [9 (link)].
First, we performed an Allplex™ GIPH assay on 17 stool samples positive for S. mansoni eggs by microscopy. After DNA extraction with the MICROLAB^® STARlet, DNA extracts were immediately collected and stored at −20 °C until amplification using the previously described primers, probe, and cycling conditions [9 (link)] with the TaqMan^® Universal PCR Master Mix reagent and the Applied Biosystems StepOnePlus™ device. As for microsporidia, DNA extracts were amplified plain and diluted to 1:10.

Six SNPs were chosen for further validation which based on the prediction scores, minor allele frequency (MAF) and function annotation (Additional file 2: Table S2). The SNP genotyping was performed using an improved multiplex ligation detection reaction (iMLDR) technique or TaqMan, which was newly developed by Genesky Biotechnologies, Inc. (Shanghai, China). We designed primers and probes for TaqMan genotyping assays for SNP rs182089527. Each genomic DNA sample (20 ng) was amplified with TaqMan universal PCR master mix reagent (Applied Biosystems, Foster City, CA) combined with the specific TaqMan SNP genotyping assay mixture, corresponding to the SNP to be genotyped. The iMLDR technique was applied for genotyping remaining SNPs which follow the instruction.
The sequencing of PDE1A designed primers as following:
Upper primer:TGACCTCTCACATATGCTGCTGT.
Lower primer:TTGGTGAGCTCTCTTGGATCA.

Diagnosis of microsporidia was based on previously described molecular methods [8 (link), 16 (link)]. Stool samples were suspended in phosphate buffered saline (PBS) and 100 μL of supernatant was added to 900 μL of ASL buffer (Qiagen, Courtaboeuf, France). After 5 min incubation at room temperature, 200 μL of supernatant was incubated for 10 min at 70 °C before DNA extraction with the EZ1^® Advanced XL device (Qiagen, Courtaboeuf, France) and the EZ1^® DSP Virus Kit (Qiagen, Courtaboeuf, France). An exogenous DNA, DiaControlDNA™ (Diagenode Diagnostics, Liège, Belgium), was added during extraction and amplified by qPCR to assess the presence of PCR inhibitors. All DNA extracts were amplified undiluted and diluted to 1:10, using the previously described primers, probe, and cycling conditions [8 (link), 16 (link)]. Reaction mix included TaqMan^® Universal PCR Master Mix reagent (Applied Biosystems France, Villebon-sur-Yvette, France) and amplification was done with the Applied Biosystems StepOnePlus™ system (Applied Biosystems France, Villebon-sur-Yvette, France). Not all patients were initially screened for microsporidia, but only those with combination of diarrhea and immunodeficiency.

We selected and genotyped nine single nucleotide polymorphisms (SNPs) at seven oxidative stress-related genes from NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/): NQO1 (rs1800566, rs10517), SOD2 (rs4880), SOD3 (rs1799895), PON1 (rs662, rs854560), GSTP1 (rs1695), GSTT1 (rs2266637), and NOS3 (rs1799983). Genomic DNA was extracted from the whole blood by the standard phenol-chloroform method with AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Scientific, Inc.). All SNPs were genotyped by the TaqMan assay method by using the ABI 7900 DNA Detection System (Applied Biosystems, Foster City, California). All probes and primers were designed by the Assay-on-Design service of Applied Biosystems. The standard polymerase chain reaction (PCR) was performed by using the Taqman Universal PCR Master Mix reagent (Applied Biosystems).

From liver biopsy specimens, a 3-mm-long sample was saved from each subject to measure gene expression [23 (link)]. Total RNA was isolated from the samples using a ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA). Reverse transcription was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to produce cDNA, according to the manufacturer’s instructions. Real-time PCR was performed using a StepOnePlus™ Sequence Detection System (Applied Biosystems) with the TaqMan Universal PCR Master Mix reagent (Applied Biosystems).
From liver tissue, we determined mRNA levels of IRS1, IRS2, β-catenin, GCK, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Target values were normalized to the expression of GAPDH.
Primers and probes for IRS1 (Hs.471508), IRS2 (Hs.442344), β-catenin (Hs.476018), and GCK (Hs.1270) were purchased from an assay-on-demand facility (Applied Biosystems) as follows: IRS1, Hs00178563_m1; IRS2, Hs00275843_s1; β-catenin, Hs00355049_m1; GCK, Hs01564555_m1; GAPDH, 4326317E (Applied Biosystems).