Total RNA was extracted using the RNeasy mini kit (Qiagen, catalog # 74106) and cDNA was made using 1500μg total RNA using Applied Biosystems High Capacity cDNA Reverse Transcriptase kit (Thermo Fisher, cat# 4368814) and quantitative PCR (qPCR) was performed using specific SYBR primers, as described above, and run on QuantStudio 3 (Thermo Fisher). Relative quantification was performed using the 2-ΔΔCt method and comparing to GAPDH or 18s.
Applied biosystems high capacity cdna reverse transcriptase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems high capacity cDNA reverse transcriptase kit is a laboratory reagent used to convert RNA into complementary DNA (cDNA) molecules. The kit contains the necessary enzymes and buffers to perform this reverse transcription process, which is a fundamental step in various molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (10 mentions)
Cfx384, by Bio-Rad (7 mentions)
Fam labeled taqman universal pcr mastermix, by Thermo Fisher Scientific (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Roche (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Primer probe set, by Thermo Fisher Scientific (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Power sybr master mix, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
13 protocols using applied biosystems high capacity cdna reverse transcriptase kit
1
Total RNA Extraction and qPCR Analysis
2
Quantifying Antioxidant Gene Expression
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Total RNA was extracted from isolated cytotrophoblast, endothelial cells (HUVEC) and placental explant tissue using the RNeasy mini kit (Qiagen, Valencia, CA) and quantified using a Nanodrop ND 1000 spectrophotometer (NanoDrop technologies Inc, Wilmington, DE) and converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit (Thermofisher) as per manufacturer guidelines. Quantitative PCR was performed using Taqman gene expression assays for: HO-1, GCLC, TXN, NQO1, VCAM, ET-1. PCR was performed on the CFX 384 (Biorad, Hercules, CA) using FAM-labeled Taqman universal PCR mastermix (Applied Biosystems) with the following run conditions: 50 oC for 2 minutes; 95 oC for 10 minutes, 95 oC for for 15 seconds, 60 oC for 1 minute (40 cycles). All data were normalized to an appropriate house-keeping gene (isolated cells normalized to: GAPDH and YWHAZ; placental tissue to: TOP1 and CYC1) as an internal control and calibrated against the average Ct of the control samples. The results were expressed as fold change relative to controls. All samples were run in triplicate.
3
qPCR Validation of NGS Genes
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Total RNA was converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit (Thermofisher). Quantitative real-time reverse-transcription polymerase chain reaction (qPCR) validation was carried out for 15 of the 23 genes identified in the NGS analysis (genes assessed were RCBTB2 and SKIL which were identified from the switchBox analysis, and 13 out of 21 genes from the regression analysis and these are listed on Table 2 ) using specific FAM-labeled Taqman primer assays and universal PCR master mix (Thermofisher) on the CFX 384 (Bio-Rad, Hercules, CA) with the following run conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 1 min (40 cycles). GUSB, YWHAZ, and B2M were used as reference housekeeping genes after validating that their expressions were not altered across samples (collection timing, location, and extraction). The comparative ΔΔCT method of analysis was used to determine relative expression. A CT value over 35 cycles was used as a threshold to deem the transcript as undetectable.
4
Analyzing Gene Expression of P. aeruginosa
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For qRT-PCR analysis, untreated and treated bacteria (1×109 CFU/mL) were exposed to either 4× MIC of AgCNTs or gen-tamicin for 4 hours at 37°C. Total RNA was purified using an RNeasy Mini Kit (Qiagen NV, Venlo, the Netherlands), and quantified using a spectrophotometer (NanoDrop 2000c; Thermo Fisher Scientific). The complementary DNA synthesis was carried out in 20 µL reaction volumes using the Applied Biosystems High-Capacity cDNA Reverse Tran-scriptase Kit (Thermo Fisher Scientific). A total of 1 µg of RNA was used to amplify the oprD, lasA, creD, lecA, lecB, rpoS, creB, rsmZ, ampD, ampP, ampG, mexT, and mexR genes of P. aeruginosa using the Applied Biosystems ViiA 7 real-time PCR system. Primer pairs for each gene are shown in Table S1 . The amplification conditions used were one cycle of initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 56°C for 25 seconds, and 72°C for 30 seconds. The relative changes in gene expression were calculated using the equation 2−ΔΔCT, where all values were normalized with respect to the 16S messenger RNA levels. Each real-time PCR assay was performed in triplicate from three independent cultures, and the results are expressed as means ± standard deviation.
5
Quantitative Transcriptional Profiling of Key Genes
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RNA was converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit (Life Technologies) or the miScript II RT kit (Qiagen), as per manufacturer guidelines.
Gene expression of GATA2, VCAM-1, ET-1, YWHAZ, Topoisomerase-1, Cytochrome c1 and GUSB, B2M (Life Technologies) were quantified by real time PCR (qRT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR master mix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles). GUSB, YWHAZ and B2M were used as housekeepers for PAXgene whole blood analyses. YWHAZ was used as a housekeeper for mRNA analyses on cells. Topoisomerase-1 and Cytochrome c1 were used as housekeepers for placental tissue. Data were analysed using the ΔΔCT method of analysis.
For microRNAs, the miScript SYBR green PCR kit was used. Housekeepers miR191, SNORD 44 and SNORD 48 were measured against miR126 and miR 221. The following conditions were used to carry out the PCR reaction-
Activation step: 95 °C for 15 mins, followed by 40 cycles of: 94 °C for 15 secs, 55 °C for 30 secs, 70 °C for 30 secs. Data were analysed using the ΔΔCT method of analysis.
Gene expression of GATA2, VCAM-1, ET-1, YWHAZ, Topoisomerase-1, Cytochrome c1 and GUSB, B2M (Life Technologies) were quantified by real time PCR (qRT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR master mix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles). GUSB, YWHAZ and B2M were used as housekeepers for PAXgene whole blood analyses. YWHAZ was used as a housekeeper for mRNA analyses on cells. Topoisomerase-1 and Cytochrome c1 were used as housekeepers for placental tissue. Data were analysed using the ΔΔCT method of analysis.
For microRNAs, the miScript SYBR green PCR kit was used. Housekeepers miR191, SNORD 44 and SNORD 48 were measured against miR126 and miR 221. The following conditions were used to carry out the PCR reaction-
Activation step: 95 °C for 15 mins, followed by 40 cycles of: 94 °C for 15 secs, 55 °C for 30 secs, 70 °C for 30 secs. Data were analysed using the ΔΔCT method of analysis.
6
Quantification of Gene Expression in HUVEC, Placenta, and Omental Artery
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RNA was extracted from primary HUVECs, placenta, and omental arteries using the RNeasy mini kit (Qiagen, Valencia, CA, USA) and quantified using the Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). We converted 0.2 μg of RNA to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase Kit (Life Technologies) as per the manufacturer’s guidelines.
The gene expression of VCAM1 (Hs01003372_m1), HO-1 (Hs01110250_m1), corin (Hs00198141_m1), NPPA (Hs00383230_g1), NPR1 (Hs00181445_m1), HUVEC housekeeper YWHAZ (Hs01122454_m1), placenta housekeepers TOP1 (Hs00243257_m1), and CYC1 (Hs00357717_m1) and omental artery housekeepers B2M (Hs00187842_m1) and Actin (Hs99999903_m1) (Life Technologies) were quantified by real-time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA, USA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 15 s; and 60 °C for 1 min (40 cycles).
The gene expression of VCAM1 (Hs01003372_m1), HO-1 (Hs01110250_m1), corin (Hs00198141_m1), NPPA (Hs00383230_g1), NPR1 (Hs00181445_m1), HUVEC housekeeper YWHAZ (Hs01122454_m1), placenta housekeepers TOP1 (Hs00243257_m1), and CYC1 (Hs00357717_m1) and omental artery housekeepers B2M (Hs00187842_m1) and Actin (Hs99999903_m1) (Life Technologies) were quantified by real-time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA, USA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 min; 95 °C for 10 min; 95 °C for 15 s; and 60 °C for 1 min (40 cycles).
7
Quantitative Analysis of Placental Gene Expression
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RNA was extracted from 20 to 30 mg of RNAlater preserved frozen human placental samples by homogenization using a RNeasy mini‐kit (Qiagen, Hilden, Germany). One μg of RNA was converted to cDNA using Applied Biosystems high capacity cDNA Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA). Taqman gene expression assays (Life Technologies) for human GDF‐15 (Assays ID: Hs00171132_m1), TOP1 (Assay ID: Hs00243257_m1), and CYC1 (Assay ID: Hs00357717_m1) were used. For comparisons between human placental samples, data were normalized to the geometric mean of 2 housekeepers; TOP1 and CYC1. qRT‐PCR was performed on the CFX 384 (Biorad, Hercules, CA) using FAM‐labeled Taqman universal PCR master mix (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles).
8
RNA Extraction and RT-qPCR for Transgene
9
Quantification of Trophoblast and HUVEC Gene Expression
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RNA was extracted from primary trophoblast and HUVECs using an RNeasy mini kit (Qiagen, Valencia, CA) and quantified using the Nanodrop ND 1000 spectrophotometer (NanoDrop technologies Inc, Wilmington, DE). 0.2 μg of RNA was converted to cDNA using Applied Biosystems high capacity cDNA reverse transcriptase kit (Life Technologies) as per manufacturer guidelines.
Gene expression of VCAM-1, ET-1, MMP 14, HO-1, Endoglin, TIMP3, NQO1, GCLC, TXN, YWHAZ and GAPDH (Life Technologies) were quantified by real time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles). SYBR RT-PCR was carried out to assess gene expressions of sFlt-1 e15a and sFlt-1 i13, YWHAZ and GAPDH. Primers were designed as previously described (Geneworks, South Australia, Australia)21 (link). RT-PCR was performed using the following run conditions: 95 °C for 20 minutes; 95 °C for 0.01 minutes, 60 °C for 20 minutes, 95 °C for 1 minute (39 cycles), melt curve 65 °C to 95 °C at 0.05 °C increments at 0.05 seconds.
Gene expression of VCAM-1, ET-1, MMP 14, HO-1, Endoglin, TIMP3, NQO1, GCLC, TXN, YWHAZ and GAPDH (Life Technologies) were quantified by real time PCR (RT-PCR) on the CFX 384 (Bio-Rad, Hercules, CA) using FAM-labeled Taqman universal PCR mastermix and its specific primer/probe set (Life Technologies) with the following run conditions: 50 °C for 2 minutes; 95 °C for 10 minutes, 95 °C for 15 seconds, 60 °C for 1 minute (40 cycles). SYBR RT-PCR was carried out to assess gene expressions of sFlt-1 e15a and sFlt-1 i13, YWHAZ and GAPDH. Primers were designed as previously described (Geneworks, South Australia, Australia)21 (link). RT-PCR was performed using the following run conditions: 95 °C for 20 minutes; 95 °C for 0.01 minutes, 60 °C for 20 minutes, 95 °C for 1 minute (39 cycles), melt curve 65 °C to 95 °C at 0.05 °C increments at 0.05 seconds.
10
Quantitative Analysis of Transgene Expression
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RNA was isolated from NHP muscle and liver samples using TRIZOL (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. 10 µg of RNA was then treated with DNase I (Roche, Basel, Switzerland) according to the manufacturer's protocol. The RNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to remove DNase prior to cDNA synthesis by reverse transcription using the Applied Biosystems High Capacity cDNA Reverse Transcriptase Kit (Life Technologies, Carlsbad, CA, USA). Real-time PCR was then performed on cDNA with primers binding to the 201Ig IA transgene with Power SYBR Master Mix for detection or primer/probe set for 18S with TaqMan Gene Expression Master Mix for detection (Life Technologies, Carlsbad, CA, USA). Relative transcript expression was determined using the ΔΔCT of each sample normalized to 18S expression.
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