Log In Sign up
The largest database of trusted experimental protocols

Sybr mastermix

Manufactured by Agilent Technologies
Visit Supplier
Sourced in Canada, United Kingdom

SYBR mastermix is a ready-to-use solution for real-time PCR applications. It contains SYBR Green I dye, necessary PCR reagents, and a stabilizing buffer.

Automatically generated - may contain errors

Lab products found in correlation

Primer express 2, by Thermo Fisher Scientific (2 mentions) Trizol, by Thermo Fisher Scientific (2 mentions) Superscript 3, by Thermo Fisher Scientific (2 mentions) Luciferase mrna, by Promega (2 mentions) Iscript cdna synthesis kit, by Bio-Rad (1 mentions)

Spelling variants of this product

Brilliant III Ultra-fast SYBR QPCR mastermix kit (2 citations) Brilliant III Ultra-Fast SYBR Green Low ROX Mastermix (2 citations) Brilliant SYBR Green II Mastermix (2 citations)

3 protocols using sybr mastermix

1

Quantitative RNA Expression Analysis

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Total RNA extraction, reverse transcription, primer design and real-time PCR were carried out as previously described [11 (link),12 (link)][15 (link),16 (link)]. RNA was extracted using TRIzol (Life Technologies, Paisley, UK). Luciferase mRNA (5ng per testis, Promega UK, Southampton, UK) was added as an external standard to each sample at the start of the RNA extraction procedure. This controls for the efficiency of RNA extraction, RNA degradation, and the reverse transcription step and allows specific transcript levels to be expressed per testis. Isolated RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript III; Life Technologies). For real-time PCR, SYBR mastermix (Brilliant II, Agilent, Amsterdam, The Netherlands) was mixed with primer (100 nM) and template in a total volume of 10μl and amplified with an Agilent MX3000 cycler. All primers were designed by PrimerExpress 2.0 (Applied Biosystems, Warrington, UK). Transcript levels were normalised relative to the luciferase external standard, generating a value of transcript expression per testis. The primers used are described in S1 Table.
Rebourcet D., Monteiro A., Cruickshanks L., Jeffery N., Smith S., Milne L., O’Shaughnessy P.J, & Smith L.B. (2019). Relationship of transcriptional markers to Leydig cell number in the mouse testis. PLoS ONE, 14(7), e0219524.
+ Open protocol
+ Expand
2

Validation of Differentially Expressed Genes

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
To validate the ten genes that were up-regulated or down-regulated in the wildtype versus nfe2 knockout model across all treatments and times, quantitative real-time PCR was conducted. Total RNA (1 μg) isolated for the RNA-Seq experiment was used to synthesize cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Using the Agilent Mx3000 qPCR system (Agilent, Santa Clara, CA), qPCR was conducted with the Brillant II SYBR Master-mix on ten candidate genes and one housekeeping gene. For each sample, triplicate technical reactions in separate wells were run containing 5 ng of cDNA. The PCR conditions were 95 °C for 10 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 60 s, and 72 °C for 60 s. Following each run, a melt curve was generated to ensure the amplification of single product. Gene-specific primers are listed in Table 1. All primers were tested for amplification efficiency using a calibration dilution curve and slope calculation approach (Rutledge and Cote, 2003 (link)). β2-Microglobulin (b2m) was chosen as a housekeeping gene due to its limited variation in expression with embryonic development and chemical exposure (McCurley and Callard, 2008 (link)). Its stability was confirmed with a One-Way ANOVA with the samples used in this study. Expression of genes was analyzed using the comparative ΔΔCT method (Livak and Schmittgen, 2001 (link)).
Williams L.M., Lago B.A., McArthur A.G., Raphenya A.R., Pray N., Saleem N., Salas S., Paulson K., Mangar R.S., Liu Y., Vo A.H, & Shavit J.A. (2016). The transcription factor, Nuclear factor, erythroid 2 (Nfe2), is a regulator of the oxidative stress response during Danio rerio development. Aquatic toxicology (Amsterdam, Netherlands), 180, 141-154.
+ Open protocol
+ Expand
3

Testis RNA Extraction and Real-Time PCR Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Testis RNA was extracted using Trizol (Life Technologies, Paisley, UK) and levels of specific mRNA species were measured by real-time PCR. Total RNA was reverse transcribed using random hexamers and Moloney murine leukaemia virus reverse transcriptase (Superscript III, Fisher Scientific UK Ltd., Loughborough, UK) as described previously [25 (link)]. To normalise the data, external standard (luciferase mRNA: Promega UK, Southampton, UK) was added to each testis at the start of the RNA extraction [26 (link)]. For real-time PCR the SYBR green method was used with SYBR mastermix (Agilent Technologies, Wokingham, UK) [27 (link)]. All primers were designed by Primer Express 2.0 (Applied Biosystems, Warrington, UK) or Primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using parameters previously described [28 (link)] and the primers used are described in Additional file 2. Transcript levels were normalised to the luciferase external standard, which generates a relative value of transcript expression per testis [29 (link)].
Soffientini U., Rebourcet D., Abel M.H., Lee S., Hamilton G., Fowler P.A., Smith L.B, & O’Shaughnessy P.J. (2017). Identification of Sertoli cell-specific transcripts in the mouse testis and the role of FSH and androgen in the control of Sertoli cell activity. BMC Genomics, 18, 972.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!