One step primescript rt pcr kit perfect real time

Qiazol lysis reagent (Qiagen, Germantown, MD, USA) was used to extract RNA from cell culture and tissue samples. Total RNA (2 µg) from each sample was reverse-transcribed into cDNA using the First Strand cDNA Synthesis Kit (MBI Fermentas, Hanover, Germany). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was conducted using a One-step PrimeScript^TM RT-PCR Kit (Perfect Real-Time; Takara Bio Inc., Tokyo, Japan) and the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The 2^−ΔΔCt method was used to calculate the relative changes in gene expression [16 (link)], and Gapdh was used as an internal control. The nucleotide sequences of the primers used for the mouse samples in the real-time RT-PCR were as follows: Cramp: forward 5′-AAT TTT CTT GAA CCG AAA GGG-3′, reverse 5′-TGT TTT CTC AGA TCC TTG GGA GC-3′; Tnf: forward 5′-ATG GCC TCC TCA TCA GTT C-3′, reverse 5′-TTG GTT TGC TAC GAC GTG-3′; Il1b: forward 5′-AAG TTG ACG GAC CCC AAA AGA T-3′, reverse 5′-TGT TGA TGT GCT GCT GCG A-3′; Il6: forward 5′-AGT TGC CTT CTT GGG ACT GA-3′, reverse 5′-TCC ACG ATT TCC CAG AGA AC-3′; Cxcl10: forward 5′-AAG TGC TGC CGT CAT TTT CT-3′, reverse 5′-GTG GCA ATG ATC TCA ACA CG-3′; Gapdh: forward 5′-TGG GCT ACA CTG AGC ACC AG-3′, reverse 5′-GGG TGT CGC TGT TGA AGT CA-3′.

The PCR assays were performed on the Rotor-Gene 3000 detection system (Corbett Research, Singapore) using One-Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (Takara). All sets of reactions were carried out in a final volume of 20 l, each containing 10 l of 2× One-Step RT-PCR Buffer III, 0.4 l of MCMVf (10 M), 0.4 l of MCMVr (10 M), 0.8 l of probe (10 M), 0.4 l of Ex Taq™ (Takara) HS (5 U/l), 0.4 l of PrimeScript™ RT Enzyme Mix II, 1.0 l of total RNA or 1.0 l of RNA transcripts, and 6.6 l of RNase-free dH₂O. Amplification reactions were performed as follows: 42°C for 5 min; 95°C for 10 s; 40 cycles of 95°C for 5 s, and 60°C for 20 s. The specificity of this TaqMan assay was evaluated using six different reactions, including water control. Using the TaqMan probe, strong fluorescent signals were detected only from reactions with samples, while the signals from four other samples along with the water control were superposed to the baseline under optimized reaction conditions. These samples can be differentiated from the four other maize samples by comparing the signals of different levels. The PCR products were analyzed further by agarose gel electrophoresis. The assay with the sample displayed the expected band of 67 bp and an unexpected faint band above the 67-bp band, whereas those of the four others did not.