Mouse brains were stored for 3 to 4 months at −80°C. Total RNA was extracted using the RNeasy Lipid Tissue Mini Kit from Qiagen (Venlo, the Netherlands). RNA concentrations and detection of potential protein impurities were measured using a NanoDrop machine (Nano Drop 1000 spectrophotometer from Thermo Scientific, Waltham, MA). The High Capacity Reverse Transcription Kit with RNase Inhibitor (Life Technologies, Grand Island, NY) was used to synthesize cDNA. Mouse primers were obtained from Life Technologies. Real Time-PCR was run using TaqMan Fast Universal PCR Master Mix, No AmpErase UNG (Life Technologies) using a 7500 Fast RT-PCR machine from Applied Biosystems (Life Technologies): 20 seconds at 95°C; 45 cycles for 3 seconds at 95°C and 30 seconds at 60°C, total volume equals 20 µl. Each target gene was measured in triplicate. HPRT1 was selected as the endogenous control after careful establishment of consistently reproducible measurements across different samples.
High capacity reverse transcription kit with rnase inhibitor
Manufactured by Thermo Fisher Scientific
The High Capacity Reverse Transcription Kit with RNase Inhibitor is a laboratory tool designed for the conversion of RNA to complementary DNA (cDNA). The kit includes reagents necessary for the reverse transcription process and an RNase inhibitor to protect the RNA from degradation during the reaction.
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Lab products found in correlation
7500 fast rt pcr machine, by Thermo Fisher Scientific (2 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (2 mentions)
Ge sample loading reagent, by Standard BioTools (2 mentions)
Dna suspension buffer, by Teknova (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (2 mentions)
96 pooled taqman assays, by Thermo Fisher Scientific (2 mentions)
Assay loading buffer, by Standard BioTools (2 mentions)
Primed 96.96 dynamic array chip, by Standard BioTools (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Universal taqman mastermix, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using high capacity reverse transcription kit with rnase inhibitor
1
Mouse Brain RNA Extraction and RT-qPCR
2
Single-Cell Gene Expression Profiling
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Total RNA was purified using the RNeasy Plus Micro Kit (Qiagen). cDNA was synthesized using all RNA available (or 1–5 ng) with the High-Capacity Reverse Transcription Kit with RNase Inhibitor (Life Technologies) (25° C for 10 min, 37° C for 120 min, 85° C for 5 min). cDNA equivalent to 1000 sorted cells was subjected to gene-specific preamplification using Taqman Preamp MasterMix (Applied Biosystems) and 96 pooled TaqMan Assays (Applied Biosystems) (Supplementary Tables 21 –23 ) at final concentration 0.2X (95°C for 10 min, followed by 16 cycles of 95°C for 15 s and 60°C for 4 min). The preamplified cDNA was diluted 5-fold in DNA suspension buffer (Teknova) and was mixed with TaqMan Universal PCR Master mix (Life Technologies) and 20X GE sample loading reagent (Fluidigm). 20X Taqman assays were diluted 1:1 with 2X assay loading buffer (Fluidigm). Taqman assays mixtures were loaded onto a primed 96.96 Dynamic Array chip (Fluidigm). The chip was loaded into the IFC Controller, where each sample was mixed with each assay in every possible combination. The chip was transferred in a Biomark (Fluidigm) for real-time PCR amplification and fluorescence acquisition using single probe (FAM-MGB, reference: ROX) settings and the default hot-start protocol with 40 cycles. Cycle thresholds (Ct) were calculated using the Fluidigm BioMark software v1.4.2.
3
RT-qPCR Validation of RNA-seq Findings
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cDNA was synthesised from 500 ng of total RNA using the High‐Capacity Reverse Transcription kit with RNase Inhibitor (Life Technologies, N8080119). Semiquantitative PCR was performed using Universal TaqMan Master Mix or Universal TaqMan Advanced Master Mix (RNA‐seq validation only) (Life Technologies, 4364340 and 4444965) on an ABI 7500 cycler or a Quant Studio 7 Pro PCR cycler (RNA‐seq validation only) with TaqMan gene expression assays (Life Technologies, 4331182): Δ40p53 [as previously described [19 (link)]] and TP53 (Hs01034249_m1). Relative expression was determined using the 2−ΔΔCt method [25 (link)] by normalising to the housekeeping gene β‐microglobulin (B2M) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) as previously described [19 (link)]. Additionally, the expression of five differentially expressed genes identified by RNA‐seq (based on mean normalised counts and relevance) was confirmed by RT‐qPCR using the following probes: LRG1 (Hs00364835_m1), HYOU1 (Hs00197328_m1), UBE2QL1 (Hs00331876_m1); SERPINA5 (Hs04333915_m1) and PCDH7 (Hs05574398_g1).
4
Single-Cell Gene Expression Profiling
5
Robust RNA Quantification from Frozen Cells
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Flash-frozen aliquots of lymphoblast cells were stored long-term (1–6 months) at -80°C. RNA was extracted using the RNeasy Mini Plus Kit from Qiagen (Venlo, Limburg). RNA concentrations and detection of potential protein impurities were measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). The High Capacity Reverse Transcription Kit with RNase Inhibitor (Life Technologies) was used to synthesize cDNA.
Human primers were obtained from Life Technologies. Real-Time PCR was run using TaqMan Fast Universal PCR Master Mix, No AmpErase UNG from Life Technologies using a 7500 Fast RT-PCR machine from Applied Biosystems (Carlsbad, CA): 20 seconds at 95°C; 40 cycles for 3 seconds at 95°C and 30 seconds at 60°C, total volume equals 20 μl. Thirty different endogenous controls were screened and carefully tested. The three (HPRT1, GAPDH, and CDKN1A) with the least amount of variation between treated and untreated samples were compared before selecting HPRT1 as the final internal control. Each target gene (Table 2 ) was measured in triplicate on each of the three flasks per treatment group.
Human primers were obtained from Life Technologies. Real-Time PCR was run using TaqMan Fast Universal PCR Master Mix, No AmpErase UNG from Life Technologies using a 7500 Fast RT-PCR machine from Applied Biosystems (Carlsbad, CA): 20 seconds at 95°C; 40 cycles for 3 seconds at 95°C and 30 seconds at 60°C, total volume equals 20 μl. Thirty different endogenous controls were screened and carefully tested. The three (HPRT1, GAPDH, and CDKN1A) with the least amount of variation between treated and untreated samples were compared before selecting HPRT1 as the final internal control. Each target gene (
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