7900 ht real time pcr system

MiRNA-enriched samples were reverse transcribed using the miRNA reverse transcription kit according to manufacturer's protocols (Life Technologies Inc., Applied Biosystems). Preamplification of miRNA cDNAs was performed by TaqMan Preamp master mix kit, and real-time PCR performed with the TaqMan Fast Universal PCR Master Mix (2X) No AmpErase UNG (Roche, Branchburg, New Jersey) using a 7900 HT Real Time PCR system. Custom designed Taqman miRNA assays were used for detection of selected novel miRNAs in real-time PCR (Life Technologies Inc., Applied Biosystems). RNU6 was used as endogenous control.
Analysis of the relative expression data was performed using the 2^−ΔΔCT method. The fold change was calculated as log₁₀ RQ where RQ is 2 ^−ΔΔCT. Log₁₀RQ correlates directly with up- (positive value) and down-regulation (negative value). A value of 1 in log₁₀RQ means a 10 fold increase in expression. Similarly, a value of -1 represents 10 fold less expression compared to control. A miRNA was considered differentially expressed if the expression level was significantly different in at least 3 out of 5 sALS spinal cords.

Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total cellular RNA, which was later prepared in cDNA by reverse transcription using the PrimeScript RT Master Mix (538100; Toyobo, Osaka, Japan). Thereafter, the Applied Biosystems 7900HT real-time PCR system (Foster City, CA, USA) was used for qPCR through SYBR-Green PCR Master Mix (15153900; Roche, Basel, Switzerland). The cDNA concentration was adjusted to 30 ng/μL. Fluorescence qPCR was performed as follows: 2 min at 95°C, then 15 s at 95°C, and 30 s at 60–68°C for 40 cycles, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being the endogenous reference gene. Fold changes (FCs) in the gene level were determined using the 2-ΔCt approach. All assays were carried out in triplicate. The primer sequences for GNAI2 were forward primer: TACCGGGCGGTTGTCTACA and reverse primer: GGGTCGGCAAAGTCGATCTG.