Total RNA from tissue samples or cells was extracted by using Trizol reagent (Invitrogen, CA). RNA was first reversely transcribed into cDNA by using RT reagent Kit (TOYOBO, Japan). Then the cDNA was subjected to RT-PCR with an SYBR Green RT-PCR Master Mix kit (TOYOBO, Japan) in an ABI PRISM 7500 system (Applied Biosystems, USA) by using the miR-126 primers set and U6 primers set (Ribobio, China). The primer sequence of miR-126 is 5′- CATTATTACTTTTGGTACGCGAAA-3′. The relative levels of miR-126 transcripts were normalized to the control U6 mRNA, and the primer sequence was 5′-TCGTGAAGCGTTCCATATTTTTAA-3′. All samples were normalized to internal controls, and the relative expression level was calculated through the 2−ΔΔCt analysis method. Experiments were performed in triplicate samples. Relative gene expression was quantified using the GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA) and expressed as a percentage of the control.
Sybr green rt pcr master mix kit
Manufactured by Toyobo
Sourced in Japan, United States
SYBR Green RT-PCR Master Mix kit is a ready-to-use solution for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. It contains SYBR Green I dye, all necessary reagents, and a thermostable reverse transcriptase and DNA polymerase enzyme system for the detection and quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rt reagent kit, by Toyobo (1 mentions)
U6 primers set, by RiboBio (1 mentions)
Abi prism 7500 system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Prism 4, by GraphPad (1 mentions)
Reverse transcription kit, by Toyobo (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Real time pcr detection system, by Eppendorf (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Rneasy mini kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnaprotect bacteria reagent, by Qiagen (1 mentions)
Pyrex, by Iwaki (1 mentions)
5 protocols using sybr green rt pcr master mix kit
1
Quantification of miR-126 Expression in Tissue and Cell Samples
2
Extracting and Quantifying RNA from Cells
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Trizol purchased from Invitrogen (CA, United States) to extract the total RNA from cell and tissue samples, and the Nano-Drop 2000 micro-spectrophotometer was used for RNA quantification. The cDNA was prepared by the reverse transcription according to the instructions of the Toyobo reverse transcription kit (Toyobo, Osaka, Japan) and amplified by SYBR Green RT-PCR Master Mix kit (Toyobo, Osaka, Japan) on the StepOne Plus system (Thermo Fisher Scientific, Waltham, MA, United States). Glyceraldehyde-3-Phosphate Dehydrogenase (Gapdh) to test the gene expression data and normalize mRNA levels of different inflammatory factors. The primer sequences used in the reaction are listed in Table 1 .
3
Quantitative Gene Expression Analysis in SH-SY5Y Cells
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Total RNA was isolated from cultured SH-SY5Y cells by using the TRIzol reagent, and a ReverTra Ace qPCR RT kit (Toyobo Co., Ltd., Osaka, Japan) was used to obtain cDNA. Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green RT-PCR Master Mix kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's protocol and then amplified with the realtime PCR detection system (Eppendorf AG, Hamburg, Germany). Amplification conditions were set as 40-cycles program (95 • C for 15 s, 60 • C for 30 s, and 72 • C for 45 s). The mRNA level of the target gene described below was normalized to that of β-actin, and the results were analyzed using the 2 -△△Ct method (Livak and Schmittgen, 2001) . Sequences of the upstream and downstream PCR primers to detect NOD2 mRNA used in qRT-PCR were 5 ′ -TGT GCG GAC TCT ACT CTT-3 ′ and 5 ′ -GTG AAC CTG AAC TTG AAC TC-3 ′ , respectively. Sequences of the upstream and downstream PCR primers to detect BACE1 mRNA were 5 ′ -TCT GTC GGA GGG AGC ATG AT-3 ′ and 5 ′ -CCA CGG AAA CTT TGT AAT GA -3 ′ , respectively. Sequences of the upstream and downstream PCR primers to detect β-actin were 5 ′ -GTG GAC ATC CGC AAA GAC-3 ′ and 5 ′ -TAG AAA GGG TGT AAC GCA ACT A-3 ′ , respectively.
4
Bacterial RNA Quantification by Real-Time RT-PCR
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Total bacterial RNA was isolated from infected hindlimbs, and then cDNA was synthesized using a Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time RT-PCR analysis was performed using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Toyobo SYBR green RT-PCR master mix kit (Toyobo Life Science, Osaka, Japan). Data for rpoB were used as the internal control. Primers are listed in Table 1 .
5
Isolation and Quantification of Streptococcus pyogenes RNA
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