Total RNA was extracted using the RNAeasy Plus RNA Extraction Kit (Qiagen). RNA was then quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and cDNA produced using the High-Capacity RNA to cDNA Kit (Thermo Fisher Scientific). Gene expression was then determined using Universal qPCR Mastermix (Thermo Fisher Scientific) with Taqman Gene Expression Assays (Thermo Fisher Scientific) on a BioRad CFX96 qPCR thermocycler. Data are normalized to mouse β ACTIN (ACTB) expression.
Cfx96 qpcr thermocycler
Manufactured by Bio-Rad
The CFX96 qPCR thermocycler is a real-time PCR detection system designed for quantitative gene expression analysis. It features a 96-well format and is capable of performing precise, sensitive, and reliable quantitative PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Rnaeasy plus rna extraction kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Amv reverse transcriptase kit, by Promega (1 mentions)
Galaxy s7 smartphone, by Samsung (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Rneasy miniprep kit, by Qiagen (1 mentions)
5 protocols using cfx96 qpcr thermocycler
1
RNA Extraction, cDNA Synthesis, and Gene Expression Analysis
2
Trizol-based Total RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from cultured plates or tumors from xenografts were lysed using Trizol (Invitrogen, Carlsbad, CA) and subjected to the Trizol Plus Total Transcriptome Isolation protocol of the PureLink RNA mini kit (Ambion - Life Technologies, Carlsbad, CA). Final RNA concentrations were determined using a BioDrop uLite spectrophotometer (Harvard BioScience, Holliston, MA). RNA was converted to cDNA using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Waltham, MA). qPCR reactions were performed using the Taqman FAST Universal PCR master mix (Applied Biosystems, Waltham, MA) in a Bio-Rad CFX96 qPCR thermocycler (Hercules, CA). U6 was used as a control for miRNA expression normalization. TaqMan assays used for miRNAs (Applied Biosystems, Waltham, MA, cat# 4427975): hsa-miR-30b, 000602; hsa-miR-200c, 002300; hsa-miR-203a, 000507; U6, 001973.
3
RNA Extraction, cDNA Synthesis, and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extract by using the TripleXtractor reagent (Bio-Cell) as indicated by the supplier, and the AMV Reverse Transcriptase kit (Promega) was used to produce cDNA following the manufacturer’s recommendations, by using 2 μg of total RNA. Quantitative PCR reactions were performed by using the Bio-Rad CFX96 qPCR thermocycler. The primer pair sequences for selected amplicons were designed using the online IDT PrimerQuest Tool software (IDT; https://eu.idtdna.com/Primerquest/Home/Index ). Primer sequences are available in Supplementary Table 1 . Gapdh mRNA level was used as an internal control and results were expressed as previously described56 (link).
4
Low-profile PCR Strip-based Fluorescence Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All experiments were performed in low-profile 0.2-mL PCR strips (Bio-Rad cat. no. TLS-0801) covered with optical flat strips (Bio-Rad cat. no. TLS-0803). Sample tubes were placed in an aluminum sample block (LightLabs cat. no. A-7079) on a hot plate (HP30A digital aluminum hotplate, Torrey Pines Scientific, Carlsbad, CA). A cardboard box large enough to cover the hot plate was painted black and two flexible cables of 96 W, 480 nm, 672 lm, 96-LEDs (DealeXtreme cat. no. 180563) were affixed to the inside top cover of the box. LEDs were powered using a single output DC power supply (UA8001A, Agilent Technologies, Santa Clara, CA). A Samsung Galaxy S7 smartphone (Samsung Electronics Co., Ltd.) was outfitted with a 520 ± 10 nm bandpass filter (Edmund Optics cat. no. 65–699) for visual detection of emitted green light (Supplementary Fig. 1). All qPCR reactions were performed on a Bio-Rad CFX96 qPCR Thermocycler.
5
Quantifying HD5 Expression in Colon Biopsies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-Time RT-PCR was used to measure transcript levels of HD5. RNA was extracted from three human colon biopsy samples each from moderate UC and CC, and diverticulitis (DV) as a non-IBD control (RNeasy Miniprep Kit, Qiagen, CA). cDNA was generated using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Pre-designed TaqMan probes (Thermo Fisher Scientific, Waltham, MA) were purchased for HD5 and GAPHD control, and all samples were run in triplicate using a CFX96 qPCR thermocycler (Bio-Rad). Data were analyzed per the ΔΔCt method of analysis.
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!