All general reagents and chemicals were purchased from Sigma-Aldrich, including angiotensin II (ANGII, A9525), unless otherwise specified. Lipofectamine RNAiMax, Bradford protein assay reagent, Trizol and SuperScript II RT were bought from Invitrogen. SiRNAs oligos were purchased from Ambion (Negative Control 4390843; HDAC3 4390771) or Invitrogen (DBC1 MSS211964 and SIRT1 MSS234959). Antibodies were purchased from Bethyl (anti DBC1, 434 A), Abcam (anti tubulin 7291, anti BrdU 6326, anti KI67 16667), or Cell Signaling (anti Cyclin D1 9262, anti PCNA 92552). DNase I and Fast SYBR Green were purchased from Roche.
Fast sybr green
Manufactured by Roche
Sourced in Switzerland
Fast SYBR Green is a real-time PCR reagent designed for rapid and sensitive detection of DNA sequences. It provides a simple and efficient method for quantifying gene expression or DNA copy number. The product is based on the SYBR Green I dye, which binds to double-stranded DNA and emits fluorescence upon binding, allowing for real-time monitoring of the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Bradford protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Superscript 2 rt, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
5 protocols using fast sybr green
1
ANGII-Induced Cell Proliferation Assay
2
Quantitative RNA Expression Analysis from Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was purified from tissues using TRIzol Reagent (Life Technologies, Carlsbad, USA) and Rneasy Kit (QIAGEN) from EWAT and liver. RNA was reverse transcribed to cDNA using a cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Semiquantitative real-time PCR analysis was performed using Fast SYBR Green (ROCHE, Basel, Switzerland). Relative expression levels were determined by normalizing each Ct value to either gene expression for mice samples using the ΔΔCt method. The primer sequences are shown in Table 1 .
3
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liver and EWAT samples were obtained from all mice. Total RNAs of liver and EWAT tissues were extracted using TRIzol Reagent (Life Technologies, Waltham, MA, USA) and a RNeasy Kit (QIAGEN, Germantown, MD, USA). The complementary DNA (cDNA) was obtained using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA samples were amplified by a quantitative reverse-transcription polymerase chain reaction using Fast SYBR Green (Roche, Basel, Switzerland). Relative expression levels were determined by normalizing each Ct value to either gene expression for mice samples using the △△Ct method. The primer sequences used in this study are shown in Table 1 .
4
Thyroid Gene Expression Analysis by qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Complementary DNA (cDNA) was synthesized from all the mice in the study (not just from the thyroids used for next-generation sequencing (NGS) using 250 ng of thyroid RNA and the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCRs were performed on StepOnePlus or ViiA 7 Real-Time PCR System instruments (Applied Biosystems, Foster City, CA, USA) using FAST SYBR green (Kapa Biosystems, Woburn, MA, USA) under the following conditions: 3 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 25 s at 60 °C. Gene expression changes were calculated on the basis of the ΔΔCt method. Standard curves were used to assess the efficiency of each reaction, and melt curve analysis was included to verify that each PCR reaction had a single product. Genes related with thyroid economy and oxidative stress response were selected randomly for gene expression analysis by real-time PCR. The primer sequences were previously reported [2 (link)].
5
RNA Isolation and Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from RNAlater-preserved tissues was isolated by using TRIzol (Invitrogen, Carlsbad, CA) and further purified by using the RNeasy mini-kit with DNAse digestion (Qiagen, Valencia, CA) as previously described (13 (link)). RNA was quantified by spectrophotometry, and the purity was assessed by the absorbance ratios of 260:280 and 260:230 nm. RNA integrity was assessed by agarose gel electrophoresis. cDNA was synthesized by using 250 ng of RNA and the SuperScript VILO cDNA Synthesis Kit (Invitrogen). cDNA was diluted 20 to 80 times, and 5 μL was used for each reaction of 15 μL final volume. Real-time PCR was performed on a ViiA 7 Real-Time PCR System instrument (Applied Biosystems, Foster City, CA) by using FAST SYBR green (Kapa Biosystems, Woburn, MA) under the following conditions: 3 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C and 25 seconds at 60°C. The primers used are shown in Supplementary Table S1 . Gene-specific amplification was confirmed by melt curve analysis. Gene expression in tissues was quantified by using the comparative delta-CT method with Ppia as the reference gene. PCR amplification efficiencies were estimated by using the LinRegPCR software (23 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!