Total RNA was extracted from mouse lung tissues, AOs, pAECs and undifferentiated hPSC cultures using an RNeasy Mini kit (Qiagen, Duesseldorf, Germany) and complementary DNA was synthesized using TOPscripTM RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a Step One Plus real-time PCR system (Applied Biosystems, Warrington, UK) with TOPrealTM qPCR 2X PreMIX (Enzynomics). All the mRNA expression was normalized to an internal control GAPDH. The primer sequences for human and mouse genes are listed in Supplementary Tables 1 and 2 , respectively.
Topscrip rt drymix
Manufactured by Enzynomics
Sourced in Germany
TOPscrip RT DryMIX is a ready-to-use reagent for reverse transcription (RT) reactions. It contains all the necessary components, including reverse transcriptase, required for the conversion of RNA to cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Topreal qpcr 2x premix, by Enzynomics (3 mentions)
Topreal qpcr 2 premix, by Enzynomics (3 mentions)
Nucleospin rna mini kit, by Macherey-Nagel (2 mentions)
Asteponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
7 protocols using topscrip rt drymix
1
Gene Expression Analysis of Mouse and Human Cells
2
HeLa Cell Total RNA Extraction and Gene Expression Analysis
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Total RNA was extracted from HeLa cells using a RNeasy Mini kit (Qiagen, Duesseldorf, Germany) and cDNA was synthesized using TOPscrip TM RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a Step One Plus real time PCR system (Applied Biosystems, Warrington, UK) with TOPreal TM qPCR 2X PreMIX (Enzynomics). The mRNA expression was normalized to an internal control GAPDH. The expression levels of the target gene mRNAs were calculated by comparing them to the expression levels of GAPDH using the 2 -ΔΔCt method. The primer sequences are listed in Table
3
Quantitative Gene Expression Analysis
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Total RNA was extracted from hPVCs, lung tissues and alveolar organoids (AOs) using an RNeasy Mini kit (Qiagen, MD, USA). cDNA was synthesized using the TOPscripTM RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a Step One Plus real-time PCR system (Applied Biosystems, Warrington, UK) with TOPrealTM qPCR 2× PreMIX (Enzynomics, Daejeon, Korea). Relative expression was normalized against GAPDH expression by the ∆∆Ct method. Sequences of primers used in this study are listed in Supplementary Materials Table S1 .
4
Quantitative RT-PCR Analysis of Liver Markers
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Total RNA was isolated from cells and transcribed into cDNA using the NucleoSpin RNA Mini kit (Macherey-Nagel GmbH & Co., Germany) and TOPscrip RT DryMIX (Enzynomics, Korea), respectively, according to the manufacturer’s instructions. Quantitative polymerase chain reaction (qPCR) was performed using TOPreal qPCR 2× PreMIX (Enzynomics) on a StepOnePlus real-time PCR system (Thermo Fisher Scientific). The relative expression was calculated by the comparative Ct (2−ΔΔCt) method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The primer sequences used were as follows: ALB, forward 5′-GACACCA TGCCTGCTGATCT-3′, reverse 5′-CACGAGAGTTGGGGTTGA CA-3′; ASGPR1, forward 5′-CCACATGGGCCCCTTAAACA-3′, reverse 5′-GGGTTTCAATACGCACCCCT-3′; GAPDH, forward 5′-CCTGCGACTTCAACAGCAAC-3′, reverse 5′-TGGGATAGG GCCTCTCTTGC-3′.
5
Lung Tissue RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted from lung tissues using an RNeasy Mini kit (Qiagen,
Duesseldorf, Germany) and cDNA was synthesized using the TOPscrip™ RT
DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a
Step One Plus real-time PCR system (Applied Biosystems, Warrington, UK) with
TOPreal™ qPCR 2X PreMIX (Enzynomics). Relative expression was normalized
against GAPDH expression by the ΔΔCt method. Sequences of primers
used in this study are listed inTable
1 .
Duesseldorf, Germany) and cDNA was synthesized using the TOPscrip™ RT
DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a
Step One Plus real-time PCR system (Applied Biosystems, Warrington, UK) with
TOPreal™ qPCR 2X PreMIX (Enzynomics). Relative expression was normalized
against GAPDH expression by the ΔΔCt method. Sequences of primers
used in this study are listed in
1
6
Bupivacaine Effects on Catabolic Markers in Chondrocytes
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We investigated the possible effects of bupivacaine on the gene expression of different catabolic markers including MMP-3, MMP-13, ADAMTS-5, and COMP. After euthanasia, total RNA was isolated from chondrocytes of the harvested joints (n = 3 for each group) and transcribed into cDNA using the NucleoSpin RNA Mini kit (Macherey-Nagel GmbH & Co., Germany) and TOPscrip RT DryMIX (Enzynomics, South Korea), respectively. Quantitative real-time polymerase chain reaction (PCR) was performed using TOPreal qPCR 2 × PreMIX (Enzynomics) on a StepOnePlus real-time PCR system (Thermo Fisher Scientific) following the recommendations of the manufacturer. The relative gene expression was calculated by the comparative Ct (2−ΔΔCt) method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. The primer sequences used for qPCR are listed in Table 1 .
7
Quantitative Gene Expression Analysis
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Total RNA was extracted from A549, AECs, AOs and undifferentiated hPSC cultures using an RNeasy Mini kit (Qiagen, Duesseldorf, Germany), and cDNA was synthesized using TOPscrip™ RT DryMIX (Enzynomics, Daejeon, Korea). PCR amplification was performed using a Step One Plus real-time PCR system (Applied Biosystems, Warrington, UK) with TOPreal™ qPCR 2X PreMIX (Enzynomics). Relative expression was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression by the ΔΔCt method. The primer sequences are listed in Table 1 .
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