Quantitative real-time PCR was performed to validate the reliability of the RNA-seq gene expression data. One microgram of RNA was reverse transcribed into cDNA using oligo(dT)18 and the TranScriba kit (A&A Biotechnology, Gdańsk, Poland). qRT-PCR reactions were performed using the Sensitive RT HS-PCR Mix (A&A Biotechnology, Gdansk, Poland) and the qTOWER3 real-time PCR thermal cycler (Analytik Jena, Jena, Germany). All TaqMan primers were purchased from Thermo Fisher Scientific/Invitrogen, Waltham, MA, USA and included in Supplementary Materials Table S4 . The relative levels of the transcripts were quantified by the 2−ΔΔCt method. GAPDH was used for standardization.
Sensitive rt hs pcr mix
Manufactured by A&A Biotechnology
Sourced in Poland
Sensitive RT HS-PCR Mix is a ready-to-use solution for reverse transcription and real-time PCR amplification. It contains all the necessary components, including a thermostable reverse transcriptase and a hot-start DNA polymerase, to perform sensitive and specific RNA detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Transcriba kit, by A&A Biotechnology (3 mentions)
Qtower 3 real time pcr thermal cycler, by Analytik Jena (2 mentions)
Total rna mini plus, by A&A Biotechnology (2 mentions)
Real time pcr thermal cycler, by Bio-Rad (1 mentions)
Taqman fast universal master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
4 protocols using sensitive rt hs pcr mix
1
Validation of RNA-seq Gene Expression
2
Quantifying DYRK1A Transcript Levels
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Total RNA was isolated using Total RNA Mini Plus (A&A Biotechnology, Gdansk, Poland, 036–100) according to the manufacturer’s protocol. RNA (1μg) was reverse-transcribed into cDNA using oligo(dT)18 and TranScriba Kit (A&A Biotechnology, Gdansk Poland, 4000–100). qRT-PCR reactions were performed using the Sensitive RT HS-PCR Mix (A&A Biotechnology, Gdansk, Poland, 2017-2000P) and the real-time PCR thermal cycler (Bio-Rad). All TaqMan primers were from Thermo Fisher Scientific/Invitrogen: DYRK1A (Mm00432934_m1), GAPDH (cat. no. 4352932E). The relative levels of the transcripts were quantified by the 2−ΔΔCt method, using GAPDH as a reference gene.
3
Reverse Transcription and qPCR Analysis
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For reverse cDNA transcription, the Transcriba reverse transcription kit was used (A&A Biotechnology, 4000–100) following the manufacturer’s instructions. The RT-qPCR was performed using Sensitive RT HS-PCR Mix (A&A Biotechnology,2017–149 2000) on the Light Cycler 480 (Roche) under the following conditions: 120 s at 50°C, the 20s at 95°C then 40 cycles at 95°C for 3 s and 60°C for 30 s. Total RNA isolated from cerebellar slices, TDAG KO and control WT mice was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). RT—qPCR was performed on the LightCycler 480 (Roche) using the TaqMan FAST Universal Mastermix (Applied Biosystems, 4304437). Cycling conditions were: 20 seconds (s) at 95°C, then 45 cycles at 95°C for 3 s, and 60°C for 30 s. The FAM dye-labelled TaqMan probes (Applied Biosystems) were used in all experiments and are listed in S2 Table . The relative mRNA expression was determined using the ΔΔCt method, calculated from absolute quantification after normalization to the endogenous housekeeping gene (β-actin).
4
Quantitative Real-Time PCR Analysis
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Total RNA was isolated using the Total RNA Mini Plus (A&A Biotechnology, Gdansk Poland) accordingly to the manufacturer’s protocol. RNA (1µg) was reverse-transcribed into cDNA using oligo(dT)18 and TranScriba Kit (A&A Biotechnology, Gdansk Poland) or using SuperScript II Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR reactions were performed using Sensitive RT HS-PCR Mix (A&A Biotechnology, Gdansk, Poland) and the qTOWER3 real-time PCR thermal cycler (Analytik Jena, Jena, Germany) or the KAPA SYBR FAST qPCR and Rotor-Gene 3000 Real-Time DNA analysis system (Corbett Research, Mortlake, Australia). Primer sequences were as follows: RIPK4 forward: 5′-ATG CCC ACT ACC ACG TCA AG-3′ and reverse 5′-TCT TCT CAT CTG CAA ACG GCT-3′; RPS17 forward 5′-AAT CTC CTG ATC CAA GGC TG-3′ and reverse 5′-CAA GAT AGC AGG TTA TGT CAC G-3′. A mathematical model including an efficiency correction was used to calculate relative expression of selected genes versus a reference gene RPS17. All TaqMan primers were from Thermo Fisher Scientific/Invitrogen: RIPK4 (Hs01062501_m1), Fibronectin-1 (Hs01549976_m1), MMP2 (Hs01548727_m1), MMP9 (Hs00957562_m1), GAPDH (cat. No. 4326317E). The relative levels of transcripts were quantified by the 2−ΔΔCt method, using GAPDH as a reference gene.
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