Quantitative reverse transcription PCR (qRT-PCR) was performed by using an Applied Biosystems Step One Plus System and Power SYBR Green PCR Master Mix (TransGen Biotech). RNA was extracted from 50 oocytes using QIAGEN RNeasy Mini Kit, and cDNA was generated by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using an ABI 7500 real-time PCR instrument and a Fast 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, United States). The sequences of all primers used are listed in Supplementary Table S1 . GAPDH was used as a reference gene. The relative expression of genes was calculated with the comparative threshold cycle (CT) method as 2−△△CT.
Step one plus system
Manufactured by Transgene
The Step One Plus System is a laboratory instrument designed for real-time PCR (polymerase chain reaction) analysis. It is capable of performing precise quantitative analysis of nucleic acid samples. The system provides accurate data on the presence and concentration of specific target sequences within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Thermo Fisher Scientific (2 mentions)
Abi 7500 real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Transgene (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Fast 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
Transcript one step gdna removal and cdna synthesis supermix, by Transgene (1 mentions)
Transstart tip green qpcr supermix, by Transgene (1 mentions)
Primer premier v5, by Premier Biosoft (1 mentions)
3 protocols using step one plus system
1
Quantitative Transcriptome Analysis of Oocytes
2
Genetic Linkage of SfMyb Expression and Bt Resistance
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To generate a BC family to determine whether reduce SfMyb expression is genetically linked with resistance, a DH-R male was crossed with a DH-S female to generate F1 progeny. Then, F1 male moths were crossed with DH-R female moths to create a BC family. The progeny from the BC family were either fed artificial diet or artificial diet amended with a diagnostic dose (0.5 μg/cm2) of Vip3Aa toxin. For 20 survivors on treated diet and 20 survivors on untreated diet, the midgut was removed and RNA was extracted from it as described above.
First-strand cDNA was synthesized using 1 μg of total RNA and TransCript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Primers were designed using Primer Premier 5 software, and qRT-PCR was performed on a StepOne Plus system with TransStart Tip Green qPCR SuperMix (TransGen Biotech) as described before (80 (link)). Relative expression levels were calculated using the 2–ΔΔCT method and normalized to the β-actin gene.
First-strand cDNA was synthesized using 1 μg of total RNA and TransCript One-step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Primers were designed using Primer Premier 5 software, and qRT-PCR was performed on a StepOne Plus system with TransStart Tip Green qPCR SuperMix (TransGen Biotech) as described before (80 (link)). Relative expression levels were calculated using the 2–ΔΔCT method and normalized to the β-actin gene.
3
Quantitative RT-qPCR protocol for oocytes
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Quantitative reverse transcription PCR (RT-qPCR) was performed by using an Applied Biosystems Step One Plus System and Power SYBR Green PCR Master Mix (TransGen Biotech). RNA was extracted from 50 to 70 oocytes using QIAGEN RNeasy Mini Kit, and cDNA was made by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). cDNA was treated with RNase H and diluted 1:10 in H2O, with 8 μl used per PCR. Gapdh was used as a control. Real-time PCR was performed as SYBR Green assays using an ABI 7500 real-time PCR instrument (Applied Biosystems). Primers used in this assay are designed using the software Primer Premier v5.0 (Premier Biosoft International) and shown in Supplementary Table 1 . Experiments were performed in biological triplicate and technical duplicate, with data represented as means ± SEM.
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