Sybr exscript rt pcr kit

Gene expression for the TLR pathway including TLR2, TLR4, MYD88, and NFκB; for the Nrf2 pathway, including Nrf2, HO-1, and NQO-1; and for apoptotic markers, including Bax and Bcl2 were quantified via real-time PCR (qPCR), consuming the primers’ sequences in compliance with the method described elsewhere [30 (link)]. Briefly, RNA was isolated and purified using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), then reverse transcribed using the reverse transcription-polymerase chain reaction (RT-PCR) kit (TaKaRa, Kusatsu, Shiga, Japan), following the manufacturer’s procedures. qPCR was applied using SYBR ExScript RT-PCR kit, and quantification examinations were completed via Opticon-2 Real-time PCR reactor (MJ Research, Capital Court, Reno, NV, USA). qPCR results were obtained using Step PE Applied Biosystems (Waltham, MA, USA) software. Target gene expressions were evaluated and correlated to the β-actin as the reference gene, and the results are shown in figures as relative expressions. The primer sequences used in this study were mentioned in Table 1.

Gene expression for the TLR pathway, HMGB-1, TLR2, TLR4, myeloid differentiation primary response gene 88 (MYD88), and NFκB were quantified via real-time PCR (qPCR) consuming the primers’ sequences, shown in Table 2, in agreement with the method described elsewhere [30 (link)]. Briefly, RNA was isolated and purified using a Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), then a reverse transcription polymerase chain reaction (RT-PCR) kit (TaKaRa, Kusatsu, Shiga, Japan) to reverse transcription reaction following the manufacturer’s procedures. qPCR was applied using a SYBR ExScript RT-PCR kit, and quantification examinations were accomplished via an Opticon-2 Real-time PCR reactor (MJ Research, Capital Court, Reno, NV, USA). qPCR results were obtained using Step PE Applied Biosystems (Waltham, MA, USA) software. Relative gene expression data were calculated as mentioned earlier by the Livak et al. (2001) [31 (link)] method (2^−ΔΔCq2) and presented as a fold change. Target gene expressions were assessed and related to the reference gene (β-actin), and the results are shown in the figures as relative expression.

The ischemic brain hemispheres were used for extracting total RNA according to the Trizol kit instructions. The MMLV-RT kit was used to reverse transcribe total RNA into cDNA. The MMP-9 primer (5′-AAATGTGGGTGTACACAGGC-3′, 3′-TTCACCCGGTTGTGGAAACT-5′), MMP-2 (5′-TGCCATCCCTGATAACCTG-3′, 3′-CAGCCAGTCCGATTTGATG-5′), Bcl-2 (5′-CCGGGAGATCGTGATGAAGT-3′, 3′-ATCCCAGCCTCCGTTAT CCT-5′), Bax (5′-GTGGTTGCCCTCTTCTACTTTG-3′, 3′-CACAAAGATGGTCACTGTC TGC-5′) and the internal reference β-actin primer (5′-ATC CTG CGT CTG GAC CTGG-3′, 5′-TTG GCA TAG AGG TCT TTA CGG AT-3′) were amplified by PCR. In a 25 μL reaction volume, PCR was performed as follows: initial denaturation for 4 min at 94 °C, followed by denaturation for 30 s at 94 °C, annealing for 30 s at 62 °C, and extension for 2 min at 72 °C for 35 cycles, and final extension for 10 min at 72 °C. qPCR was applied using an SYBR ExScript RT-PCR kit, and quantification examinations were accomplished via an Opticon-2 Real-time PCR reactor (MJ Research, Capital Court, Reno, NV, USA). qPCR results were obtained using Step PE Applied Biosystems (Waltham, MA, USA) software. Relative gene expression data were calculated using the (2^−∆∆Cq2) method and presented as a fold change [72 (link)]. Target gene expressions were assessed and related to the reference gene, and the results were presented in the figures as relative expressions.