7900ht fast real time pcr system software

RNA was isolated by using PureLink RNA Mini Kit (Thermo-Fisher) following manufacturer’s recommendations. One to two micrograms of RNA were reverse transcribed in a 20 μl reaction using oligo(dT)^{20 (link)} and SuperScript III First-Strand Synthesis System (Thermo-Fisher). Relative quantitative real-time PCR was performed in a total reaction volume of 10 μl, using 2 μl (20 μg/μl) cDNA, 1 μl gene specific primer mix (QuantiTect primer Assays), 5 μl SYBR GreenER qPCR SuperMix (Thermo-Fisher), and 2 μl water. The quantification of gene expression was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems) in triplicate. The thermal profile of the reaction was: 50 °C for 2 min, 95 °C for 10 min and 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Amplification of the sequence of interest was normalized with a reference endogenous gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The value was expressed as a fold change relative to RNA from cells infected with control adenovirus (Ad. Null), control siRNA (siControl), or non-treated cells. For data analysis, the 7900HT Fast Real-Time PCR System Software was used (Applied Biosystems).

DNA extraction and SNP genotyping was performed according to the manufacturer’s instructions in the laboratories and under the supervision of the Department of Clinical Pharmacology, University Medical Center Goettingen, Germany.
Whole blood samples were drawn from all study subjects within 72 h after sepsis onset. The extraction of genomic DNA was performed using either the QIAmp^® DNA Blood Kit in QIAcube^®, the EZ1^® DNA Blood Kit in BioRobot EZ1^® or the AllPrep DNA Mini Kit (all from Qiagen, Hilden, Germany), as previously described [21 (link),24 (link),43 (link)]. Quantity and quality of the extracted DNA were tested by spectrophotometric measurement.
The LAG-3 rs951818 was genotyped in all samples through TaqMan polymerase chain reaction (PCR) using the appropriate predesigned TaqMan^® SNP Genotyping Assay C___8921385_10 (Thermo Fisher Scientific, Waltham, MA, USA) and a 7900HT Fast-Real-Time PCR System (Life Technologies, Darmstadt, Germany) as well as 7900HT Fast-Real-Time PCR System software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, CA, USA). Over 20% of the samples were genotyped in duplicate to increase reliability.

For the current study, 125 matched samples were provided by the Department of Pathology of the First Affiliated Hospital of Guangxi Medical University. Formalin fixation and paraffin embedding (FFPE) were performed to preserve the specimens. This aspect of the research was authorized by the hospital’s ethics committee. Next, the miR-144-3p expression in 125 paired clinical samples was detected by RT-qPCR with the Applied Biosystems 7900HT Fast Real-Time PCR System software. The miR-144-3p sequence was as follows: UACAGUAUAGAUGAUGUACU. The formula for 2-Δcq was used for the calculation of the miR-144-3p expression value.