CN, NL, and GA were supplied by Mavi Sud, Aprilia (LT, Italy). Chloroform (code: 102442), 2-butanol (code: 109630), lithium bromide (LiBr2), acetic acid (code: 33209), ethanol (EtOH), poly(ethylene glycol) (PEG8000), and Dulbecco’s phosphate-buffered saline (DPBS) were provided by Sigma-Aldrich (Milan, Italy). Immortalized human keratinocytes, HaCaT cell line, were obtained from ATCC-LGC Standards (Milan, Italy). MgCl2, Dulbecco’s Modified Essential Medium (DMEM), L-glutamine, penicillin, streptomycin and fetal calf serum were purchased from Invitrogen, (Carlsbad, CA, USA). Alamar Blue was bought from Thermo Fisher Scientific (Waltham, MA, USA). LC Fast Start DNA Master SYBR Green kit was obtained from Roche Applied Science (Euroclone S.p.A., Pero, Italy).
Lc fast start dna master sybr green kit
Manufactured by Roche
Sourced in Switzerland
The LC Fast Start DNA Master SYBR Green kit is a reagent used for real-time PCR analysis. It contains all necessary components, including a DNA polymerase and SYBR Green dye, to enable fast and efficient DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Oligonucleotide primers, by TIB Molbiol (2 mentions)
Nucleospin rna kit, by Macherey-Nagel (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified essential medium dmem, by Thermo Fisher Scientific (1 mentions)
Polyethylene glycol peg 8000, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Merck Group (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
2 butanol, by Merck Group (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Rotor gene 6000 system, by Qiagen (1 mentions)
Rneasy total rna kit, by Qiagen (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Lightcycler, by Roche (1 mentions)
6 protocols using lc fast start dna master sybr green kit
1
Cytotoxicity Evaluation of Novel Compounds
2
Transcriptional Analysis of Skin MC Response to TSLP
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Skin MCs (5 × 105 cells/mL) were deprived of GF/serum for 16 h in minimal medium. After starvation, cells were incubated with TSLP for the indicated time points. RT-qPCR was performed as described, using primers described therein [14 (link)]. Briefly, total RNA was isolated using the Nucleo spin RNA Kit (Macherey-Nagel, Düren, Germany), and RT-qPCR was carried out with the Light Cycler (LC) Fast Start DNA Master SYBR® Green kit (Roche Applied-Science, Basel, Switzerland).
The expression levels of the target gene were quantified relative to the expression of the reference gene Cyclophilin B, using the 2−ΔΔCT method. The oligonucleotide primers (TIB Molbiol, Berlin, Germany) for SCF and IL-33 were as follows:
The expression levels of the target gene were quantified relative to the expression of the reference gene Cyclophilin B, using the 2−ΔΔCT method. The oligonucleotide primers (TIB Molbiol, Berlin, Germany) for SCF and IL-33 were as follows:
3
Gastrointestinal Microbial DNA Extraction and Quantification
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DNA was extracted from gastrointestinal tract contents or fecal samples of rats using the NucleoSpin Soil kit protocol (Macherey-Nagel SARL, Hoerdt, France). DNA was amplified with LC-FastStart DNA Master SYBR green kit (Roche Diagnostics, France) and specific primers BL18SPPF1 (5’-AGTAGTCATACGCTCGTCTCAAA-3’ ) and BL18SR2PP (5’-TCTTCGTTACCCGTTACTGC-3’ ) using a Rotor-Gene 6000 system (Corbett Life Science, France) as described by Poirier et al [10 (link)].
4
Quantifying MRGPRX2 Expression via RT-qPCR
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Total RNA was isolated using the RNeasy Total RNA Kit, digested with RNase free DNase (Qiagen, Hilden, Germany), and PCR carried out with the LC Fast Start DNA Master SYBR Green kit (Roche Applied Science). Primers for MRGPRX2 were 5′-GGATCAGGAAGACCGGGATCA and 5′-CGGCCTGGGGAACAGAAAGT. The values were normalized to the housekeeping genes β actin, cyclophilin B, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), each ratio contrasted against control conditions (set as 1) and the mean of the three determinations was used for the analysis and is depicted in the figures [30 (link),31 (link),33 (link)].
5
Quantitative Gene Expression Analysis
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Total RNA was extracted following the Chomczynski method [12 (link)]. Reverse transcription of mRNA was carried out according to the manufacturer’s instructions. Gene expression levels were compared among subjects by the comparative threshold cycle (ΔΔCT) quantitation method. We used a LightCycler and an LC Fast Start DNA Master SYBR Green Kit (Roche Applied Science). AQP1, AQP2, AQP3, eNOS, iNOS and IL-6 gene expression were analysed using primer pairs described in the Supplementary data , Table S2 . Real-time amplification of the Snail and E-cadherin gene fragments was performed with primers and conditions previously described [13 (link)]. Normalization was performed against a 147-bp amplified fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The normalized SNAIL/E-CADHERIN mRNA ratio was used as a marker of epithelial-to-mesenchymal differentiation as described [14 (link)]. Depicted data represent the difference between the second and first PETs (PET2–PET1). Polymerase chain reaction product amplification efficiencies were obtained from device software and by plotting CT differences against pooled cDNA diluted ranging from 1 to 1/100, by confirming that slopes were less than specificities analysed by melting curve analysis and also confirmed by agarose gel electrophoresis.
6
Quantifying gene expression by RT-qPCR
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RT-qPCR was performed as described.38 (link) In
brief, total RNA was isolated using the Nucleo spin RNA Kit (Macherey-Nagel, Düren,
Germany), and RT-qPCR was carried out with the LC Fast Start DNA Master SYBR Green kit
(Roche Applied-Science, Basel, Switzerland). The oligonucleotide primers (TIB Molbiol,
Berlin, Germany) were as follows:The expression levels of the target gene were quantified relative to the expression of
the reference gene β-actin using the 2-ΔΔCT method.
brief, total RNA was isolated using the Nucleo spin RNA Kit (Macherey-Nagel, Düren,
Germany), and RT-qPCR was carried out with the LC Fast Start DNA Master SYBR Green kit
(Roche Applied-Science, Basel, Switzerland). The oligonucleotide primers (TIB Molbiol,
Berlin, Germany) were as follows:
the reference gene β-actin using the 2-ΔΔCT method.
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