Total RNA was extracted from human TPSs collected from culture plates after 8 weeks of culture, using RNeasy® Mini Purification Kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions. Reverse transcription was performed using the SuperScript VILO™ complementary DNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed using a StepOne™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) and TaqMan® probes were used for TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA): human CCL1 (Hs00171072-m1), CCL2 (Hs00234140-m1), CCL3 (Hs00234142-m1), CCL4 (Hs99999148-m1), CCL5 (Hs00982282-m1), SDF-1 (Hs03676656_mH), and human glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1).
Superscript vilo complementary dna synthesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Superscript VILO complementary DNA synthesis kit is a reagent system used for the production of complementary DNA (cDNA) from ribonucleic acid (RNA) samples. The kit provides the necessary components, including reverse transcriptase enzyme, buffer, and reaction mix, to enable the conversion of RNA into its DNA counterpart for subsequent analysis or applications.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Abi 7900 pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman probe chemistries, by Thermo Fisher Scientific (2 mentions)
Picopure, by Thermo Fisher Scientific (2 mentions)
Rneasy mini purification kit, by Qiagen (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Magna pure lc robot, by Roche (1 mentions)
Magna pure lc total nucleic acid isolation kit, by Roche (1 mentions)
Quantstudio 7 flex real time polymerase chain reaction system, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Aurum total rna mini kit, by Bio-Rad (1 mentions)
Sybr green reaction master mix, by Bio-Rad (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Isogene, by Nippon Gene (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Magmax 96 total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
9 protocols using superscript vilo complementary dna synthesis kit
1
Quantifying Gene Expression in Human TPS Cultures
2
Serum Nucleic Acid Extraction and cDNA Synthesis
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Blood was incubated in an upright position at room temperature for 30–45 minutes to allow clotting. Then the sample was centrifuged for 15 minutes at 2000 relative centrifugal force to obtain serum. Total nucleic acid was extracted from serum samples using the automated Roche MagNA Pure LC robot and the MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics, Indianapolis, IN), and eluted with 50 μL of elution buffer according to the manufacturer's instructions. Complementary DNA was generated using the high-temperature capability SuperScript VILO complementary DNA Synthesis Kit (Invitrogen, Life Technologies, Carlsbad, CA) on an ABI PRISM19700 PCR system (Thermo Fisher Scientific, Waltham, MA).
3
Quantitative Gene Expression Analysis of Colon Organoids
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The cells of colonic organoids, day 3 chips, and day 7 Colon Chips were harvested with RLT buffer from the RNeasy Mini Kit (74106; Qiagen, Hilden, Germany). RNA was extracted using the RNeasy Mini Kit (74106; Qiagen) followed by complementary DNA synthesis with the SuperScript VILO complementary DNA Synthesis Kit (1754250; Invitrogen). Reverse-transcription polymerase chain reaction was performed using TaqMan Fast Advanced Master Mix (4444965; Applied Biosystems, Foster City, CA), TaqMan Gene Expression Assays (Hs02786624_g1 for glyceraldehyde-3-phosphate dehydrogenase, Hs00358836_m1 for Kruppel-like factor 4, Hs00171942_m1 for SAM pointed domain ETS factor, Hs00356521_m1 for anterior gradient 2, protein disulfide isomerase family member, Hs00395669_m1 for resistin-like molecule β, Hs00175398_m1 for Fc-γ binding protein, Hs01086545_m1 for Kallikrein 1, Hs00983260_m1 for Meprin A subunit β, and Hs00976287_m1 for chloride channel accessory 1; Thermo Fisher Scientific), and run on a QuantStudio 7 Flex Real-Time polymerase chain reaction System (Applied Biosystems). All results were normalized relative to glyceraldehyde-3-phosphate dehydrogenase expression and day 0 organoid respective gene expression.
4
Quantitative RT-qPCR for Gene Expression
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Total RNA was extracted using Aurum total RNA mini kit (Bio‐Rad) according to manufacturer’s instructions. One microgram total RNA was used for complementary DNA synthesis using the Superscript VILO complementary DNA synthesis kit (Thermo Fisher Scientific), and reaction mixture was incubated using a C1000 touch thermal cycler (Bio‐Rad) as follows: 25 °C for 10 minutes, 37 °C for 2 hours and 85 °C for 5 minutes. The reaction mixture was further diluted to 300 and 2 µL complementary DNA as the template was subjected to quantitative reverse transcription–polymerase chain reaction, which was performed in triplicate for each gene using a SYBR Green reaction master mix (Bio‐Rad). Real‐time polymerase chain reaction conditions included initial denaturation step at 95 °C for 10 minutes, 40 cycles of 2‐steps with 15 seconds of denaturation at 95 °C, followed by 1 minutes of annealing at 60 °C using Applied Biosystems 7500 real‐time polymerase chain reaction systems. The messenger RNA levels of the genes examined were normalized to GAPDH messenger RNA levels. The primers used for the genes are listed in Table 1 .
5
Quantitative gene expression analysis in Caco-2 and human tissues
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Total RNA was isolated from Caco-2 cells, human iPSC–derived cells using ISOGENE (NIPPON GENE, Tokyo, Japan). Total RNA of Human Adult Normal Tissue 5 Donor Pool: Small Intestine and Human Adult Normal Tissue: Colon was purchased from BioChain Institute. Complementary DNA was synthesized using 500 ng of total RNA with a Superscript VILO complementary DNA synthesis kit (Thermo Fisher Scientific). Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) using a StepOnePlus real-time PCR system (Applied Biosystems). The relative quantitation of target messenger RNA levels was performed by using the 2-ΔΔCT method. The values were normalized by those of the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR primer sequences were obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/ ).
6
RNA Isolation and qRT-PCR Analysis
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Briefly, RNA isolation was performed using the MagMAX-96 total RNA isolation kit (AM1830; Ambion, Waltham, MA). SuperScript VILO complementary DNA synthesis kit (11754250; ThermoFisher) was used to make complementary DNA from 200 ng RNA. Complementary DNA levels were detected using QuantiTect SYBR Green (608056; Qiagen). Relative gene expression was plotted as arbitrary units, using the following formula: [2^(housekeeping gene Ct - gene Ct)] × 10,000. All primer sequences are listed in Supplementary Table 3 . The Ct values for all of the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) experiments are provided in Supplementary Table 4 .
7
RNA Isolation and Quantification Protocol
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Cells were washed in cold PBS and harvested using the TriPure Isolation Reagent (Sigma–Aldrich Co; catalog no.: 11667157001). RNA samples were purified using the GeneJET RNA Purification Kit (Thermo Fisher Scientific; catalog no.: K0732) and quantified using a NanoDrop 2000 spectrophotometer prior to reverse transcription using the SuperScript VILO Master Mix kit (Thermo Fisher Scientific; catalog no.: 11755500). For nascent transcripts, the Click-iT Nascent RNA Capture Kit (Thermo Fisher Scientific; catalog no.: C10365) was used. Briefly, cells were treated with 0.5 mM 5-ethynyl-2′-deoxyuridine for the indicated times and washed in cold PBS before RNA extraction. After biotinylation, nascent RNA molecules were captured using streptavidin beads and used as a template for complementary DNA synthesis using the SuperScript VILO complementary DNA synthesis kit (Thermo Fisher Scientific; catalog no.: 11754-050). Real-time qPCR was performed using the SYBR Select Master Mix (Thermo Fisher Scientific; catalog no.: 4472920). Primer sequences for each gene are listed in Table S1 .
8
Quantitative Real-Time PCR Tissue Analysis
9
Quantitative Real-Time PCR Tissue Analysis
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Adhesive patches were macrodissected to separate lesional tissue from surrounding normal tissue. Total RNA was isolated from the recovered lesional tissue using a modified PicoPure procedure (Life Technologies, Foster City, CA) and reverse transcribed to complementary DNA using SuperScript VILO complementary DNA synthesis kits (Thermo Fisher Scientific, Pittsburgh, PA). The resulting complementary DNA was subsequently used for target gene expression analysis with quantitative real-time (qRT)-polymerase chain reaction (PCR) on an ABI7900 PCR system (Life Technologies). Each qRT-PCR reaction used 3 pg of total RNA, in duplicate, in 20-μL volume on 384-well PCR reaction plates using predesigned gene-specific TaqMan probe chemistries (Life Technologies). An averaged cycle threshold value of the duplicate measurements was used in the analysis. Gene expression was considered detected if the quantitative polymerase chain reaction yielded an amplification curve and a measurable cycle threshold value, or not detected if the reaction yielded an undetermined cycle threshold value (amplification curve never above detection threshold). In addition to the 2 target genes, human β-actin was used as an internal control.
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