Total RNA was isolated from nematodes using Trizol (Invitrogen, UK) according to manufacturer’s protocols. RNAs were first assessed for their purity and concentration by OD260/280 in a spectrophotometer, and then used for cDNA synthesis performed in a 12.5 μL reaction volume containing 625 ng total RNA, 0.5 mM reverse-transcript primers, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 units ribonuclease inhibitor and 100 U reverse transcriptase (Takara, China). After cDNA synthesis, relative expression levels of examined genes were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). Relative quantification of targeted genes in comparison to reference tba-1 gene encoding tubulin was determined. The final results were expressed as relative expression ratio between the targeted gene and the reference gene. Primer information was shown in Table S7 . All reactions were performed in triplicate.
7500 real time pcr system
Manufactured by Biotium
Sourced in United States
The 7500 real-time PCR system is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qPCR) analysis. It is capable of performing real-time detection and quantification of nucleic acid sequences.
Automatically generated - may contain errors
5 protocols using 7500 real time pcr system
1
Quantifying Gene Expression in Nematodes
2
Nematode RNA Isolation and Gene Expression
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Total RNA of nematodes was isolated using Trizol (Invitrogen, UK) according to the manufacturer’s protocols. After cDNA synthesis, relative transcriptional expressions of certain genes were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). All reactions were performed in triplicate with the same cDNA samples. Relative quantification of the examined genes in comparison to reference tba-1 gene encoding a tubulin protein was determined. The primer information was shown in Table S3 .
3
Nematode Total RNA Isolation and qPCR Analysis
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Total RNA of nematodes was isolated using the reagent of Trizol (Invitrogen, UK) according to the manufacturer’s protocol. Purity and concentration of the isolated RNAs were evaluated by a ratio of OD260/280 using a spectrophotometer. After the cDNA synthesis, transcriptional expressions of the examined genes were determined by real-time PCR in an ABI 7500 real-time PCR system with Evagreen (Biotium, USA). Relative quantification of targeted genes was expressed as transcriptional expression ratio between the examined genes and the reference gene of tba-1 encoding alpha-tubulin protein, pmp-3 encoding a putative ABC transporter, or act-1 encoding an actin. All the reactions were performed in triplicate, and the replicates are biological. Primer information for real-time PCR of the examined genes and the reference gene was shown in Table S6 .
4
RNA Extraction and qRT-PCR Analysis in C. elegans
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Total RNA was extracted from C. elegans according to the manufacturer’s protocol in an RNeasy Mini Kit (Qiagen). Purity and concentration of RNAs were analyzed by the ratio of OD 260/280 in a spectrophotometer. cDNA was synthesized in a 12.5 μl reaction volume containing 625 ng total RNA, 0.5 mM reverse-transcript primers, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 units of ribonuclease inhibitor, and 100 units of reverse transcriptase (Takara, China). The reaction mixture was incubated at 25°C for 5 min, followed by 42°C for 60 min. The reverse transcriptase was inactivated at 70°C for 15 min. Transcriptional quantification was determined by real-time PCR in an ABI 7500 real-time PCR system using Evagreen (Biotium, USA). The putative antimicrobial genes were selected as described [18 (link)]. The final results were expressed as relative expression ratio between targeted genes including the antimicrobial genes and reference tba-1 gene encoding a tubulin protein. All reactions were performed in triplicate. The related primers for targeted genes and reference gene were shown in Table S1 .
5
Quantitative Analysis of Nematode miRNAs
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Total RNA was extracted from nematodes according to the manufacturer’s protocol in RNeasy Mini Kit (Qiagen). Purity and concentration of RNAs were analyzed by OD 260/280 in a spectrophotometer. cDNA was synthesized in a 12.5 μL reaction volume containing 625 ng total RNA, 0.5 mM reverse-transcript primers, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 20 units of ribonuclease inhibitor, and 100 units of reverse transcriptase (Takara, China). The reaction mixture was first incubated at 25 °C for 5 min and 42 °C for 60 min. Again, the reverse transcriptase was inactivated at 70 °C for 15 min. Transcriptional quantification was determined by real-time PCR in an ABI 7500 real-time PCR system using Evagreen (Biotium, USA). Primer information for miRNAs was shown in Tables S7 and 8 . The miRNA expressions were expressed as the relative expression ratio between certain miRNA and F35C11.9 encoding a small nuclear RNA U6. The related information for antimicrobial genes was shown in Table S9 . The final results for antimicrobial genes were expressed as relative expression ratio between targeted genes and reference act-1 gene.
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