cDNAs were prepared from 100 ng of RNA using Fluidigm Reverse Transcription Master Mix (Fluidigm PN 100‐647297). Following a pre‐amplification step (Fluidigm® PreAmp Master Mix and DELTAgene™ Assay kits) and exonuclease I treatment, samples diluted in Eva‐Green® Supermix with Low ROX were loaded with primer reaction mixes in 96.96 Dynamic Array™ IFCs. Gene expression was then assessed on a Fluidigm BioMark HD instrument. Data were analyzed with real‐time PCR analysis software (Fluidigm Corporation) and presented as relative gene expression according to the ΔΔCt method. Heatmaps depicting fold changes of gene expression were prepared using MeV software.
Fluidigm reverse transcription master mix
Manufactured by Standard BioTools
Sourced in United States
Fluidigm Reverse Transcription Master Mix is a laboratory reagent designed for the reverse transcription of RNA into complementary DNA (cDNA). It contains the necessary enzymes, buffers, and other components required to efficiently perform this process.
Automatically generated - may contain errors
Lab products found in correlation
Preamp master mix, by Standard BioTools (3 mentions)
Biomark hd system, by Standard BioTools (3 mentions)
Fluidigm preamp master mix, by Standard BioTools (2 mentions)
96.96 dynamic array ifc, by Standard BioTools (2 mentions)
Usingnucleospin rna xs kit, by Macherey-Nagel (2 mentions)
Biomark hd, by Standard BioTools (1 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Evagreen supermix, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
Cfx maestro 1, by Bio-Rad (1 mentions)
Cfx96 touch system, by Bio-Rad (1 mentions)
Nucleospin rna xs kit, by Macherey-Nagel (1 mentions)
8 protocols using fluidigm reverse transcription master mix
1
Gene Expression Analysis by Fluidigm BioMark HD
2
SARS-CoV-2 and Other Coronavirus Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole pathogen control panels were purchased from Randox Laboratories Ltd, including MERS-CoV (catalogue no. QAV154181), CoV-OC43, NL63 (catalogue no. QAV164189), and SARS-CoV-2 (catalogue no. SCV2QC). Samples were extracted using the QIAamp Viral RNA Mini Kits (catalogue no. 52906). Viral nucleic acid was extracted using the manufacturer-recommended protocol49 . Viral RNA was reverse transcribed to cDNA using Fluidigm reverse transcription master mix (catalogue no. SKU 100–6299). Viral cDNA was further pre-amplified using Fluidigm Preamp master mix (catalogue no. PN 100-5744). Reverse transcription and pre-amplification were conducted according to the Fluidigm manufacturer’s protocol (Fluidigm document number: 101-7571 A2 and 100-5876 C2).
3
Zebrafish Larvae Transcriptomics After EDC Exposure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression levels in whole zebrafish larvae exposed to EDCs during 48 h (3 to 5 dpf) were assessed using the Fluidigm-BioMark HD system. Briefly, for mRNA extraction, 30 whole zebrafish larvae were pooled and homogenized in 100 μL TRIzol reagent, and total RNA was extracted according to the manufacturer’s protocol with TRIzol reagent (Gibco, Carlsbad, CA, USA). RNA samples were then reverse-transcribed and cDNAs were obtained using the Fluidigm reverse transcription Master Mix, and were then preamplified for 12 cycles in the presence of the Fluidigm Pre-Amp Master Mix. Gene expression was then measured with the Biorad EvaGreen SuperMix on a 96.96 Dynamic Array IFC. After quality control verification, mRNA expression was calculated with the ΔΔCt method and normalized by using actb2, 18 s, and gapdh as reference. Sequences of the tested zebrafish primers and official gene names are provided in Supplementary Table S1 (information about the primer conditions is available upon request).
4
RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated using Invitrogen PureLink RNA Mini Kit (Thermo Fisher Scientific) or the RNeasy Mini Kit (Qiagen) using the manufacturers’ protocol. RNA was quantified using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific). One μl of RNA was transformed into complementary DNA using Fluidigm Reverse Transcription Master Mix (Fluidigm) according to the manufacturer’s protocol and underwent 16 preamplification cycles using the Fluidigm Preamp Master Mix according to the manufacturer’s protocol. RNA was then analyzed by the UNC Advanced Analytics Core facility using the Fluidigm Biomark HD 96.96 IFC array (Fluidigm) and validated TaqMan probes according to the manufacturer’s protocol. Using BioMark HD software (Fluidigm, Table 1 ), Ct values were then normalized against the geometric mean of GAPDH and beta-actin (ACTB), and relative log2fold changes were calculated, normalizing to day 0 hTSCs based on the ΔΔCT method.
5
Isolation and cDNA Synthesis from Enriched Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from enriched cell fractions using the RNEasy® Micro Kit (Qiagen, Germany) according to the manufacturer’s instructions and stored at -80°C. The RNA purity, quality and quantity were assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies Inc.) An RNA integrity number (RIN) score above 7 was considered sufficient for downstream processing. Isolated RNA was thawed on ice and cDNA synthesized using the Fluidigm Reverse Transcription Master Mix (Fluidigm PN 100-6297) and a conventional thermal cycler (Life Technologies, United States of America). Aliquots of the cDNA were further subjected to either 16 or 20 pre-amplification cycles using the Fluidigm One Tube PreAmp Master Mix (Fluidigm PN 100-5580) and DNA was removed by exonuclease-I treatment (PN M0293S/L; 20 U/µl).
6
RNA Isolation and Quantitative PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For whole embryos, RNA was isolated from 10 embryos per sample using NucleoSpin RNA kit (Macherey‐Nagel, Duren, Germany), and total mRNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). For RNA isolation from FAC‐sorted macrophages and osteoclasts, the NucleoSpin RNA XS kit (Macherey‐Nagel) was used. RNA isolated from three independent biological samples was converted into cDNA and preamplified using Fluidigm Reverse Transcription Master Mix and Preamp Master Mix, respectively (Fluidigm, South San Francisco, CA, USA). All steps were performed following the manufacturers' standard protocols. PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) was used for qPCR conducted in a CFX96 Touch system (Bio‐Rad Laboratories, Hercules, CA, USA). Data analysis was performed using Bio‐Rad's CFX Maestro 1.0 software with β‐actin as the loading control for normalization. Two‐tailed Student's t tests were performed for statistical analysis.
7
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted usingNucleospin® RNA XS kit (Macherey-Nagel, Duren, Germany). cDNA was then generated using Fluidigm Reverse Transcription Master Mix (Fluidigm). The qPCR were performed in triplicate using 96.96 Dynamic Array™ IFCs and the BioMark™ HD System from Fluidigm. For each sample, the mean CT value for the gene of interest was calculated, normalized to the geometric mean value of the 2 housekeeping genes (CDKN1B and ELF1) (Table S3
8
Quantitative Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted usingNucleospin® RNA XS kit (Macherey-Nagel, Duren, Germany). cDNA was then generated using Fluidigm Reverse Transcription Master Mix (Fluidigm). The qPCR were performed in triplicate using 96.96 Dynamic Array™ IFCs and the BioMark™ HD System from Fluidigm. For each sample, the mean CT value for the gene of interest was calculated, normalized to the geometric mean value of the 2 housekeeping genes (CDKN1B, and ELF1) (Table S3), and compared to the median value obtained from the reference population (HD cMO or iMO for Figure 2, and DLBCL ncMO for Figure 3) using the 2-ddCT method. Results
were expressed as the ratio of sample mean to reference mean for each gene.
were expressed as the ratio of sample mean to reference mean for each gene.
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!