Rotor gene 3000 real time pcr system

Fresh spleen specimens were immediately removed from the mice mentioned above (n = 5-8 per group) at the time of sacrifice, snap-frozen in liquid nitrogen, and then were stored at -70°C until processed. Total RNA was extracted with TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using a PrimeScript^TM RT Reagent Kit (Takara Biotechnology Co., Dalian, China) according to the manufacturer's instructions. Real-time quantitative PCR analysis was performed using a Rotor-Gene 3000 Real-time PCR System (Corbett Research Inc., Mortlake NSW, Australia) with SYBR^® Premix Ex Taq^TM II (Takara) and primers specific for murine T-box transcription factor 21 (T-bet), GATA-binding protein 3 (GATA3), retinoic acid-related orphan nuclear receptor γt (RORγt), forkhead box protein 3 (Foxp3), interferon (IFN)-γ, interleukin (IL)-4, IL-17A, IL-21, and transforming growth factor (TGF)-β. These are typical cytokines and specific transcription factors to T helper (Th) cells, including Th1 (T-bet and IFN-γ), Th2 (GATA3 and IL-4) and Th17 (RORγt, IL-17A and IL-21), and regulatory T cells (Tregs; Foxp3 and TGF-β), respectively. Primers were designed and synthesized by Takara (Table 1). The transcript levels were normalized to that of murine GAPDH, and expressed as mean ± standard deviation (SD) of △Ct values.

Total RNA was extracted using the E.Z.N.A. Total RNA Kit (Omega Bio TEK) from cells. The yield and purity of the extracted RNA was measured by the A260/A280 ratio (NanoDrop, Thermo Scientific). qScript cDNA Synthesis kit was used for making the cDNA (Complementary DNA). Rotor Gene 3000 Real- time PCR system (Corbett Research) was used for performing the Real-time quantitative PCR. Briefly, the reaction mixture contained cDNA (1:10 dilution), SYBR Green PCR Master Mix (Qiagen) and 1μM of primers (Forward+ Reverse). 2^-ΔΔCT equation was used to analyze the results and was expressed as relative m-RNA expression. The following primer sequence were used; forward: 5′-AAGCCTGTAGCCCATGTTGT-3′ and reverse: 5′-GAGGTA-CAGGCCCTCTGATG-3′ for TNF-α and forward: 5′-GGATTTGGTCGTATTGGG-3′ and reverse: 5′-GGAAGATGGTGATGGGATT-3′ for GAPDH.

Total RNA was extracted from soybean and Arabidopsis plants using the RNAsolve reagent (OMEGA Bio-Tek, Norcross, GA, USA), and was then purified with RNase-free DNase I (Invitrogen, USA). About 1 μg of RNA was reversely transcribed to complementary DNA (cDNA) with MMLV-reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed using a SYBR Premix kit (Promega, Madison, WI, USA) on a Rotor-Gene 3000 real-time PCR system (Corbett Research, Mortlake, Australia). The GmEF-1a and AtEF-1a were separately used as an internal control. Relative expression of genes was calculated as the ratio of expression of the tested genes to those of EF-1a as described previously [56 (link)]. Specific gene primers are listed in Table S1.

Total RNA was extracted from the blood samples/cells of the IPF patients by using TRIzol reagent from Invitrogen (Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Complementary DNA was synthesized by using M-MLV reverse transcriptase kit from Promega (Madison, WI, USA) in accordance with the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green PCR Master Mix kit from Takara Bio (Shiga, Japan) on a Rotor Gene3000 real-time PCR system from Corbett Research (Sydney, Australia). The following PCR conditions were set: initial denaturation at 95 °C for 5 min, 30 cycles at 60 °C for 25 s, annealing at 52 °C for 20 s, and extension at 72 °C for 20 s. Fluorescence signal was monitored at 585 nm during each extension. Glyceraldehyde 3-phosphate dehydrogenase served as an internal control.

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quantity and quality were measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Complementary DNA synthesis was performed with the M-MLV reverse transcriptase kit (Invitrogen) following the manufacturer's instructions. qRT-PCR was performed with a SYBR green-based PCR master mix kit (Takara, Shiga, Japan) on a Rotor Gene 3000 real-time PCR system from Corbett Research (Sydney, Australia). The PCR conditions were as follows: initial denaturation at 95°C for 30 sec. followed by 40 cycles of 95°C for 5 sec., and 60°C for 20 sec. Fluorescence signal was monitored at 585 nm during each extension. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 served as an internal control.

To measure the binding efficiency of aptamers, 2 μM of candidate aptamers were mixed with 0.5 μg/mL of the spike protein, followed by incubation at 4 °C for 30 min. The spike protein bound to the DNA aptamers was incubated with Ni–NTA magnetic nanobeads and the conjugate was separated using a magnet, with the supernatant removed. The recovered beads were then washed five times with a binding buffer, and the aptamers were released from the nanobeads using an elution buffer. The candidate aptamers were detected using TOPreal™ SYBR Green qPCR PreMix (Enzynomics, Daejeon, Korea) to obtain the standard curve for real-time PCR analysis. Cycle threshold (Ct) values of the candidate aptamers were compared using a Rotor-Gene 3000 real-time PCR system (Corbett Research, Australia). All analyses were performed in triplicate and Ct values were calculated from the average of each measurement.