Quant one step rt pcr kit

The collected cells were prepared for total RNA extraction using using Trizol reagent (Invitrogen, USA). The cDNAs were synthesized using a reverse transcription kit (Quant One Step RT-PCR kit, TIANGEN, China), and real-time PCR was performed using SYBR green master mix (Quant one step qRT-PCR Kit, TIANGEN, China) on a MyiQTM2 (BIORAD, USA). β-actin was used to normalize the real-time PCR data. The following primer sequences were used: CTGF, 5′- GGCCTCTTCTGCGATTTCG-3'and 5′-GGCCTCTTCTGCGATTTCG-3′; EGFR, 5′-GGCCTCTTCTGCGATTTCG-3′ and 5′-GCAGCTTGACCCTTCTCGG-3′;β-actin, 5′-ATGGAGGGGAATACAGCCC-3′ and 5′-TTCTTTGCAGCTCCTTCGTT-3′.

Samples were obtained from apparently healthy calves and diseased calves for disease confirmation and serotyping. The samples were analyzed by the conventional reverse-transcriptase polymerase chain reaction (RT-PCR) method. Total RNA was extracted from EDTA-mixed whole blood using TRIzol Reagent (TRIzol™ Plus RNA Purification Kit; catalog number 12183555) following the manufacturer’s instructions. After RNA was extracted from the blood, RT-PCR was performed for serotype identification of FMDV using the specific primers described in Table-1[13 (link)-15 (link)]. For the detection of FMDV, the RNA from 20 samples was subjected to RT-PCR by TIANGEN, Quant One-Step RT-PCR Kit; catalog number KR113, according to the manufacturer’s instructions. Each One-Step RT-PCR mixture tube (50 µL) contained a different serotype primer for each separate reaction. The pan-serotype RT-PCR cycling protocol was similar, except that the annealing temperature was modified to 55°C with UTR RT-PCR. Thermocycler G-Storm was used for the detection of FMDV by RT-PCR. The PCR products were analyzed by electrophoresis through 1.5% agarose gel containing ethidium bromide (0.5 μg/mL), and the image of the amplified product was captured using a gel documentation system (Biospectrum UVP, UK).

The total RNAs were extracted from cells using the RNApure Tissue & Cell Kit (DNase I) in accordance with the manufacturer’s instructions (CWBio, Beijing, China). Then, a total of 1 μg RNA from each sample was subjected to cDNA reverse transcription and real-time PCR (RT-PCR) using the Quant One Step RT-PCR kit (TIANGEN, Beijing, China) on Bio-Rad detection system (Bio-Rad, Hercules, CA) after RNA quantification via using an ND-1000 NanoDrop Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Delaware). GAPDH expression level serves as an internal reference. Primers were listed as follows,
CD47: forward (F) 5′-CGGCGTGTATACCAATGC-3′; Reverse (R) 5′-TTTGAATGCATTAAGGGGTTCCT-3′;
GAPDH: F 5′-CCACTAGGCGCTCACTGTTCTC-3′; R 5′-ACTCCGACCTTCACCTTCCC-3′.

Total RNA was directly extracted from clinical specimens using a QIAamp viral RNA mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was eluted in a final volume of 60 μL elution buffer and used immediately or stored at −80°C. RNA from each sample was examined by conventional RT-PCR or real-time RT-PCR.
Clinical specimens collected from 2008 to 2009 were examined by conventional RT-PCR. Conventional RT-PCR was conducted using a Quant One Step RT-PCR Kit (Tiangen Biotech, Beijing, China) containing specific primers for EV71, CA16, and pan-enterovirus, respectively. The clinical specimens collected from 2010 to 2013 were examined using commercially available real-time RT-PCR kits (Diagnostic Kit for Human Enterovirus, EV71 and CA16, Beijing Kinghawk Pharmaceutical Co., Ltd, Beijing, China or Jiangshu Shuoshi Biological Technology Co., Ltd, Taizhou, China) as per the manufacturer's protocols. Test results were classified into four categories: enterovirus negative, EV71 positive, CA16 positive, or positive for another enterovirus without further serotype identification.

RT-PCR was performed using the Quant One Step RT-PCR kit (Tiangen Biotech, Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The thermal cycler parameters were: 4 min at 94°C followed by 30 cycles of 30 sec at 94°C, 30 sec at 58°C, 40 sec at 72°C and then 10 min at 72°C. The following specific primers were used: NR1, forward 5′-GCTGCACGCCTTTATCTG-3′ and reverse 5′-TCCTACGGGCATCCTTGT-3′; NR2b, forward 5′-CACGGTGCCTTCAGAGTT-3′ and reverse 5′-CCTCCTCCAAGGTGACAA-3′. The PCR products were separated using electrophoresis on a 2.0% agarose gel. The intensity of the bands was analyzed using BioSense SC-810 Gel Documentation System (Shanghai Bio-Tech Co., Ltd., Shanghai, China) and Gel-Pro 3.1 software (Media Cybernetics, Inc., Bethesda, MD, USA).

The collected cells and implanted bones were collected for total RNA extraction with Trizol reagent (Invitrogen). The complementary DNAs were synthesized with reverse transcription kits (Quant One Step RT‐PCR kit; TIANGEN), and real‐time PCR was performed with SYBR green master mix (Quant one step qRT‐PCR Kit; TIANGEN) on a MyiQTM2 instrument (Bio‐Rad). β‐Actin was used to normalize the real‐time PCR data. The primer sequences are listed in Table 1.