The corneal tissue and mouse peritoneal primary macrophages were obtained and stored at −80°C. The total RNA of the isolated cells and tissues were extracted using RNAiso plus (TaKaRa, Dalian, China) and quantified using spectrophotometry. The first-strand cDNA was synthesized through the reverse transcription of 2 µg of total RNA. The product of cDNA was diluted with diethylpyrocarbonate-treated water. The reverse-transcription polymerase chain reaction (RT-PCR) Master Mix (Takara, Dalian, China) along with SYBR Green and specific primers were used to perform the RT-PCR analysis. The cycle parameters of the reactions were as follows: 95°C for 30 seconds, then 40 cycles of 95°C for 5 seconds, followed by 60°C for 30 seconds, with the final steps at 95°C for 15 seconds, 60°C for 30 seconds, and 95°C for 15 seconds. The quantitative expression of β-actin was used as the internal control. The primers used are given in Table .
Rt pcr master mix
Manufactured by Takara Bio
Sourced in China
The RT-PCR Master Mix is a complete solution for reverse transcription and real-time PCR. It contains all the necessary reagents, including a reverse transcriptase enzyme and a DNA polymerase, to perform sensitive and reproducible gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso plus, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 instrument 2, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Takara Bio (1 mentions)
Lightcycler480ii, by Takara Bio (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using rt pcr master mix
1
Quantitative Expression of β-actin in Corneal Tissue and Macrophages
2
RT-qPCR Analysis of SMPDL3A Gene
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After extracting the total RNA using TRIzol Reagent (Invitrogen), the total RNA was reverse transcribed into cDNA using Takara RT-PCR Master Mix (DRR0036A). The RT-qPCR system, LightCycler 480 Instrument II and SYBR Green solution (Takara) were used to detect the relative expression of the target gene, SMPDL3A using β-actin as an internal reference. The primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd., China, with the following primer sequences: SMPDL3A forward primer: 5′-CTCACAGAGACAGCATTATGGTT-3′; SMPDL3A reverse primer: 5′-TTCACTGGTGTAACAGCAGGA-3′; β-actin forward primer: 5′-GGCGGCACCACCATGTACCCT-3′; β-actin reverse primer: 5′-AGGGGCCGGACTCGTCATACT-3′.
3
Quantitative Analysis of URM1 Expression
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Total cellular RNA was extracted with TRIzol (Invitrogen) and reverse-transcribed into cDNA using RT-PCR Master Mix (DRR0036A, TaKaRa) kit. The relative expression of the target gene was determined using SYBR Green (TaKaRa) with a real-time quantitative PCR system (LightCycler 480II). The primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd. The primer sequences are as follows:
URM1 forward: 3’-AAGAAACATCGAGTCACTTTGC-5’
URM1 reverse: 5’-GGTAGTCCAGCTCACCCAGTA-3’
β-actin forward: 3’-GGCGGCACCACCATGTACCCT-5’
β-actin reverse: 5’-AGGGGCCGGACTCGTCATACT-3’
URM1 forward: 3’-AAGAAACATCGAGTCACTTTGC-5’
URM1 reverse: 5’-GGTAGTCCAGCTCACCCAGTA-3’
β-actin forward: 3’-GGCGGCACCACCATGTACCCT-5’
β-actin reverse: 5’-AGGGGCCGGACTCGTCATACT-3’
4
Real-Time qPCR for Gene Expression Validation
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Quantitative Real Time (qRT) PCR was performed by using SYBR Green PCR master mix kit in a stepOnePlus Real Time PCR system (Applied Biosystem by Life Technologies, USA). To validate the gene expression profiles identified by RNA-Seq. 2 μg of RNA was reverse transcribed in a 20 μL volume with RT PCR master mix (TaKaRa) as per the manual instruction. Six gene (SaMTPS, SaFPPS, SaDSX, SaGGPS, SaGPS, and SaCYP450) specific primers were predicted using by the online tool Primer3 version 0.4.0 and synthesised at (Eurofins India Pvt. Ltd). The sequence of primers with a melting temperature between 60-61 0 C and a PCR product range of 151-229 bp were listed in S2 Table . Actin was used as a reference gene. qRT-PCR was performed with step One Real time PCR system (Applied Biosystems, Thermofisher Scientific). The qRT-PCR reaction systems were as follows: 95 0 C for 20 s, followed by 40 cycles of 95 0 C for 5 s, 60 0 C for 30s and 72 0 C 40 sec. The fluorescence data were collected and analysed with Step One analysis software.
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