The cofilin gene of S. scabiei, identified from NCBI within an expressed sequence tag (EST) (GenBank: BG817660), was amplified with primers which were designed using Primer 5.0 Software (forward primer: 5ʹ-AAATGGCCTCAGGTGTAACT-3ʹ; reverse primer: 5ʹ-GGTGGGT2233GAGATAATTTAGTTTC-3ʹ) (Invitrogen, Beijing, China). The PCR cycling conditions were: 94 °C for 5 min, 30 cycles of amplification at 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, followed by a final extension step at 72 °C for 30 min. After extraction and purification using a QIAquick Gel Extraction Kit, PCR products were cloned into vector pMD19-T (TaKaRa Bio Co. Ltd., Dalian, China). Then the plasmid was transformed into Escherichia coli strain DH5a (Fermentas) and sequenced by Invitrogen (Beijing, China). The amplified DNA sequence was analyzed and compared with the EST of S. scabiei cofilin using DNAMAN Software and compared against the NCBI database utilizing BLAST (http://www.ncbi.nlm.nih.gov/ ).
Primer 5
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Primer 5.0 software is a tool for designing and evaluating primers and probes for PCR and other nucleic acid-based applications. It provides functionality for primer design, specificity checking, and melting temperature calculation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Abi stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Abi prism 7000 sequence detection system and software, by Thermo Fisher Scientific (1 mentions)
Rq1 rnase free dnase 1, by Promega (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast qpcr master mix 2x kit, by Roche (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Reverse transcription system a3500, by Promega (1 mentions)
Abi prism 7300, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Ultraviolet spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
22 protocols using primer 5
1
Molecular Characterization of Scabies Cofilin
2
Quantitative PCR Analysis of nsp11 Gene
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For PCR, cellular DNA was extracted from MARC-nsp11 cells using QIAamp DNA kit (Qiagen) and PCR was performed to determine the nsp11 gene integration. For reverse transcription (RT), total cellular RNA was extracted using Trizol (Invitrogen) and was treated with RQ1 RNase-free DNase I (Promega) followed by RT using the nsp11-specific reverse primer and PCR using the primer set as described above. Quantitative (q) PCR was performed using ABI Prism 7000 Sequence Detection System and Software (Applied Biosystems) in a final volume of 25 μL containing 2.5 μL of cDNA synthesized from the RT reaction, 2.5 pmol of each primer, 12.5 μL of SYBR Green Master Mix (Applied Biosystems), and 5 μL of water. The primer sequences were designed using Primer 5.0 Software (Invitrogen) or obtained from previous reports (Table 2 ). The amplification parameters were 40 cycles of two steps, each step comprised of heating at 95°C and extension at 60°C. The final mRNA levels of target genes were normalized using GAPDH as a house keeping gene.
3
Quantifying Aortic Valve Gene Expression
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Specimens from aortic valves were cut into pieces and smashed with liquid nitrogen. Total RNAs from leaflets were prepared using Trizol reagent (Thermo Fisher, Waltham, MA, USA). The supernatants containing total RNAs were then purified using Direct-zol RNA MicroPrep Kit (Zymo Research). The concentration of total RNAs was measured using NanoDrop (Thermo Fisher, Waltham, MA, USA) and 0.1–1 µg of total RNAs were reverse transcribed with cDNA Synthesis Kit (Thermo Fisher, Waltham, MA, USA) in accordance with the manufacturers’ instruction. The cDNAs were further sixfold diluted and subjected into RT-qPCR experiments using QuantStudio 6 Real-Time PCR Detection System (Thermo Fisher; Waltham, MA, USA) and KAPA SYBR FAST qPCR Master Mix (2X) Kit (Kapa Biosystems, Wilmington, DE, USA). Primers for RT-qPCR were designed using Primer 5.0 software and synthesized by Invitrogen. Expression was normalized to GAPDH. The data were analyzed using the 2-ΔΔCT method.
4
Quantification of PKM2 mRNA Expression
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The total RNA was extracted using a kit (Solarbio, Shanghai, China), and then the OD260/280 of the RNA sample was detected by ultraviolet spectrophotometery. In addition, the RNA concentration was measured. The sample was then stored at -80°C for further use. Based on the Genebank data for primer sequences, Primer 5.0 software was used for PKM2 primer sequences (Invitrogen Inc., Shanghai, China) (Table 1). Reverse transcription was conducted following the instructions of Reverse Transcription System A3500 (Promega Corporation, Madison, WI, USA). Reaction conditions were as follows: initial denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min; and finally 72°C for 5 min. The reaction system contained 12.5 μL of Premix Ex Taq or SYBR Green Mix, 1 μL of Forward Primer, 1 μL of Reverse Primer, 4 μL of DNA template and 25 μL of ddH 2 O. Using GAPDH as the internal reference, a solubility curve was used to detect PCR reliability and primer specificity.
The 2 -△△Ct method was employed to assess PKM2 mRNA expression with a multiplication rate equal to the relative expression of the target gene with △Ct = Ct target gene -Ct internal reference gene [19] . This method was also adapted for the expression of PKM2, p53 and p21 after transfection.
The 2 -△△Ct method was employed to assess PKM2 mRNA expression with a multiplication rate equal to the relative expression of the target gene with △Ct = Ct target gene -Ct internal reference gene [19] . This method was also adapted for the expression of PKM2, p53 and p21 after transfection.
5
Analyzing ECM Gene Expression in IVD
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Total RNA from the NP was analyzed for the expression of ECM genes. Briefly, the AF was cut with a blade, and the NP tissue was removed using a micro curette and immediately frozen in liquid nitrogen at each harvesting time point. Tissues from 3 IVDs were pooled to ensure that a sufficient quantity of tissues was obtained for RNA isolation [28 (link), 30 (link), 31 (link)], which was performed using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Primers for the rabbit GAPDH and ECM genes were custom designed using Primer 5.0 software (Applied Biosystems, Foster City, CA) (Table 1 ).
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Green One-Step qRT-PCR kit (KAPA Biosystems, USA). The final qRT-PCR results were obtained using the comparative Ct method with the following equation: where −ΔΔCt = (Cttarget − Ctreference)control − (Cttarget − Ctreference)culture. (reference: mean Ct of GAPDH; control: day 0; and culture: day 3, 7, 14, or 21).
Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a SYBR Green One-Step qRT-PCR kit (KAPA Biosystems, USA). The final qRT-PCR results were obtained using the comparative Ct method with the following equation: where −ΔΔCt = (Cttarget − Ctreference)control − (Cttarget − Ctreference)culture. (reference: mean Ct of GAPDH; control: day 0; and culture: day 3, 7, 14, or 21).
6
Quantitative RT-PCR Analysis of VEGFA in Rabbit VEPs
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The VEPs (n = 4 per group) were collected at each time point. Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, US) was used following the manufacturer’s instructions. The mixtures were placed on ice for sonication at 30% amplitude. Each sample was sonicated for 3 s and then paused for 2 s for a total sonication time of approximately 30 s. Subsequently, chloroform, isopropanol, and alcohol were added one by one to the samples for total RNA extraction. The RNA concentration was then determined. Total RNA was reverse-transcribed into cDNA using a reverse transcription (RT) kit. PCR amplification was performed using the first-strand cDNA as the template. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 20 s, followed by a final extension at 72°C for 10 min. The PCR products were separated with agarose gel electrophoresis. Primers for the rabbit GAPDH and VEGFA genes were custom designed using Primer 5.0 software (Applied Biosystems, Foster City, CA, USA) [Table 1 ]. Grey values and relative intensity were determined and calculated using a gel imaging system.
7
Gastric Mucosal Cell RNA Extraction and Analysis
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Total RNA was extracted from gastric mucosal cells using TRIzol reagent and reverse-transcribed to cDNA using TOYOBO reverse transcription kits with cDNA Eraser, according to the manufacturer’s instructions. Then cDNA was synthesized using polymerase chain reaction system (Applied Biosystems co., Ltd., Waltham, MA, USA) at 30°C for 10min, 42°C for 60min, 99°C for 5min, 4°C for 5min. The full-length mRNA sequences of bcl-2, P53, C-MYC and NF-κB, were obtained from GenBank (http://www.ncbi.nlm.nih.gov/nucleotide ), the sequence of primers were designed by using Primer 5.0 software (Applied Biosystems co., Ltd., Waltham, MA, USA) and synthesized by Beijing Dingguo Changsheng biotechnology co., Ltd. (Beijing, China) (see Table 1 ). Primers were used for a total of 35 cycles of PCR under the following parameters: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, extension at 72°C for 10 min. PCR product quality was monitored by post-PCR melt curve analysis. Each PCR product was subjected to electrophoresis on 2% agarose gel for 30 min, then ethidium bromide-stained bands were scanned and analysed.
8
qRT-PCR Analysis of Nerve Growth Factor
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Total RNA was prepared from the peri-infracted border with Trizol reagent (Gibco, USA), and reversely transcribed to cDNA using TaqMan Reverse Transcription Reagents. The expression levels of candidate genes were measured by real-time quantitative RT-PCR using an SYBR Green PCR Master mix. In each assay, both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the nerve growth factor (NGF) gene from the same samples were amplified in triplicate in separate tubes. The mRNA levels of NGF were calculated using the relative standard curve method and normalized against the corresponding GAPDH mRNA level, and then expressed as a relative change over the control ± standard deviation (SD). The expected size amplicons were confirmed by gel electrophoreses. The sequences of the genes studied were obtained from GenBank, and the primers were designed using the PRIMER 5.0 software (Applied Biosystems, Foster City, CA, USA). The primer sequence and amplicon size of the genes are shown in Table 1 .
Primer sequence and amplicon size of genes
| Gene | Accession no. | Primer and probe sequence | Amplicon size, bp |
|---|---|---|---|
| NGF | NM_001194950.1 | F: 5′ AGA CCC GCA ACA TCA CTG TGG 3′ | 172 |
| R: 5′ GAA GAC CGC TTG CTC CTG TGA 3′ | |||
| β-Actin | NM_001195845.1 | F: 5′ ACG GGC AGG TCA TCA CTA TTG 3′ | 166 |
| R: 5′ AGC ACT GTG TTG GCA TAG AGG 3′ |
9
IL-10 Gene Expression Analysis
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The cultured cells from each group were used to isolate total RNA using TRIzol from TAKARA Biotechnology, (Dalian, China). The expression of IL10 gene was measured using a real-time PCR system, ABI Prism 7300 from Applied Biosystems (USA) and SYBR Green from TAKARA Biotechnology, the IL 10 gene expression was measured. Specific primers were used to amplify each target gene from the cDNA. The program for real-time qPCR (Quantitative reverse transcription polymerase chain reaction) was: 95 °C for 10 min, 35 cycles for 15 s at 95 °C, and for 31 s at 60 °C, and then a dissolution curve was included. The Primer 5.0 software (Applied Biosystems) was used to design primers and their respective sequences are: IFN-α2b forward, 5′-TCCAAAAGGCTGAAACCATCC-3′ and reverse, 5′-GACAACCTCCCAGGCACAAG-3'. To control the variations in the expression levels the data for expression were normalized with the housekeeping gene β-actin geometric mean, amplified using the primers: forward, 5′ACGGCAAGTTCAACGGCACAG-3′ and reverse, 5′-GACGCCAGTAGACTCCACGACA-3′. The comparison of efficiency of amplification between the reference and the target was done using the ΔΔCt calculation.
10
RNA Extraction and qRT-PCR Analysis
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Total RNA from DCMECs was extracted with TRIzol reagent (Sigma). The concentration and mass of RNA were measured at 260/280 nm using an ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA with OD 260/ 280 1•8-2•0 was reverse-transcribed to cDNA using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology, Tokyo, Japan). qRT-PCR primers were designed using Primer 5.0 software (Applied Biosystems, Foster City, CA, USA; sequences are given in online Supplementary Table S1). qRT-PCR was performed using the Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and SYBR Premix Ex Taq (TaKaRa Biotechnology). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene (Wu et al. 2015) . The relative expression level of genes was calculated by normalizing to GAPDH using the 2 -ΔΔCt method (Huang et al. 2012; Li et al. 2014) .
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