RNA extraction and cDNA synthesis were performed using the Cells-to-SignalTM kit (Life Technologies, Carlsbad, USA) as directed. mRNA levels of genes were obtained from standard curves and corrected using GAPDH or 18S ribosomal RNA levels following TaqMan PCR according to the manufacturer’s instructions (Applied Biosystems, Warrington, UK). Oligonucleotide primers were designed using Primer Express software version 1.0 (Applied Biosystems), to be in different exons close to, or spanning, an exon boundary so as to prevent amplification of residual cDNA. The sequences of primers and probes used were as previously described [80 (link)], or: GAPDH: For, 5’-GTGAACCATGAGAAGTATGACAAC-3’; Rev, 5’-CATGAGTCCTTCCACGATACC-3’; Probe, 5’-CCTCAAGATCATCAGCAATGCCTCCTG-3’; TGFBR1: For, 5’-GCAGACTTAGGACTGGCAGTAAG-3’; Rev, 5’-AGAACTTCAGGGGCCATGT-3’; Universal Probe Library (Roche, Burgess Hill, UK) Probe #5; ACVRL1: For, 5’-AGACCCCCACCATCCCTA-3’; Rev, 5’-CGCATCATCTGAGCTAGGC-3’; Probe #71; ID1: For, 5’-CCAGAACCGCAAGGTGAG-3’; Rev 5’-GGTCCCTGATGTAGTCGATGA-3’; Probe #39; PAI1: For, 5’-AAGGCACCTCTGAGAACTTCA-3’; Rev, 5’-CCCAGGACTAGGCAGGTG-3’; Probe #19. The relative gene expression in samples was then determined using either a Lightcycler (Roche Diagnostics, Indianapolis, USA) or a 7900HT PCR system (Applied Biosystems).
Primer express software version 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Primer Express software version 1.5 is a tool for designing and analyzing real-time PCR primers and probes. It provides a user interface for inputting DNA sequences and generating suitable primer and probe combinations for real-time PCR experiments.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Primescript rt pcr kit, by Takara Bio (2 mentions)
Rnaiso reagent, by Takara Bio (2 mentions)
7900ht pcr system, by Thermo Fisher Scientific (1 mentions)
Universal probe library, by Roche (1 mentions)
Lightcycler, by Roche (1 mentions)
Abi prism 7700 sequence detection system and software, by Thermo Fisher Scientific (1 mentions)
Stepone version 2, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus instrument, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Abi 9700, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green pcr mix, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Abi prism 7500, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
9 protocols using primer express software version 1
1
RNA Extraction and qPCR Analysis
2
Real-Time PCR for T-cell Receptor Excision Circles
3
RNA Extraction and qPCR Analysis
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Total RNA was extracted from cell lysates harvested in Buffer RLT Pluss using an RNeasy® Plus Kit according to the manufacturer’s protocol or from tissue homogenates by chloroform phase separation and subsequent EconoSpin column purification (Epoch Life Science). RNA was quantified using Nanodrop™ (Thermo Scientific, Waltham, MA, USA) and stored at −80 °C until analysis. Complementary DNA (cDNA) was synthesized from 2 µg mRNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) and ProFlex™ 3 × 32-well thermal cycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until analysis. Primers (Supplementary Table S1 ) were designed by the Primer Express software version 1 (Applied Biosystems, Thermo Fisher Scientific). Gene expressions were examined by real-time quantitative PCR (qPCR) using Power SYBR™ Green PCR Master Mix and the Applied Biosystems StepOnePlus™ Instrument with the accompanying software StepOne™ Version 2.3 (Applied Biosystems, Thermo Fisher Scientific). Gene expression was normalised against the geometric means of the CT values of housekeeping controls (RPLP0, GAPDH, β-actin, 18s) [30 (link)].
4
RNA Isolation and qPCR Quantification
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Isolation of RNA was performed using the RNeasy Mini Kit from Qiagen (Hilden, Germany). RNA was treated with DNase (Ambion, Austin, TX) to make sure no DNA was left to contaminate, and RNA was eluted in 30 ml RNase free H 2 O. Isolated RNA was stored at À80 C until further analysis. cDNA synthesis was carried out using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantification of mRNA was performed using qPCR Master Mix for SYBR Green I (Applied Power Master Mix) and the ABI 9700 (Applied Biosystem) with the accompanying software SDS 2.3. Primers were designed with the use of primer express software version 1 (Applied Biosystems), and primer sequences can be given upon request. Gene expression of the housekeeping gene GADPH was used for normalization.
5
Quantitative RT-PCR Analysis of Gene Expression
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As previously reported [35 (link)], RT-qPCR was used. Using TRIzol reagent, total RNA was extracted from brain tissues (Invitrogen, Carlsbad, USA). To obtain cDNA, the PrimeScript® RT Reagent Kit (Takara, Kyoto, Japan) was utilized. RT-qPCR was then performed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, USA) with a Light Cycler 96 (Roche) and SYBR Green PCR MIX (Takara). Primer Express software version 1.5 (Applied Biosystems) was used to construct the primers, and the sequences are presented in Additional file 5 : Table S2. The 2−ΔΔCt technique was used to analyze gene expression, and the relative gene expression levels were adjusted to β-actin.
6
Real-Time PCR for Gene Expression
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Real-time quantitative PCR was performed as described previously56 (link)57 (link). Total RNA for real-time quantitative RT-PCR was isolated from tissues using RNAiso Reagent (TaKaRa, Tokyo, Japan) and reverse transcripted into the single strand cDNA. Primers were designed using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA, USA). The primers for TNF-α are as follows: sense, 5′-TTCTGTCTACTGAACTTCGGGGTGATCGGTCC-3′; antisense, 5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′. The primers for IL-6 are as follows: sense, 5′-ATGGATGCTACCAAACTGGAT-3′ and antisense, 5′-TGAAGGACTCTGGCTTTGTCT-3′. Gene expression of GAPDH was used for control. The primers for GAPDH are as follows: forward, 5′-TCTGACGTGCCGCCTGGAG-3′; reverse, 5′-TCGCAGGAGACAACCTGGTC-3′. Quantification of mRNA was performed using the ABI Prism 7500 (Applied Biosystems) with PrimeScript™ RT-PCR Kit (TaKaRa, Tokyo, Japan).
7
MATR3 and NF-κB Expression Analysis
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Total RNA was extracted from the BM samples of the enrolled patients and MATR3-knockdown (KD) THP1 cells using Trizol (Invitrogen, Life Technologies, Waltham, MA, USA). MATR3 mRNA and NF-κB mRNA expression were evaluated by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and the specific forward and reverse primers for the TaqMan® probe were designed using Primer Express software version 1.5 (Applied Biosystems, Life Technologies). β-actin was used as an internal control.
8
Quantitative RT-PCR Analysis of Circadian Clock Genes
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A quantitative reverse transcription polymerase chain reaction (QRT-PCR) was applied extensively in genomics studies, especially where target-specific primers and real-time technology served a greater advantage [47 (link)]. By using the TRIzol reagent, the total RNA was isolated from the SH-SY5Y cells (Invitrogen, Camarillo, CA, USA) following the manufacturer’s instructions. The reverse transcription was performed using the cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany). The cDNA sequence of the three circadian clock genes (PER1, CLOCK, BMAL1) and SIRT1 were evaluated, and the specific forward and reverse primers and MGB TaqMan® probe were designed using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA, USA). Polymerase chain reactions (PCRs) were performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
9
RNA Isolation and qRT-PCR Quantification
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Total RNA for real-time quantitative RT-PCR was isolated from tissues using RNAiso Reagent (TaKaRa, Tokyo, Japan) and reverse transcripted into the single strand cDNA. Primers were designed using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA, USA) (Additional file 2 : Table S2). Quantification of mRNA was performed using the Thermo Q50 (Thermo Fisher Scientific) with PrimeScript™ RT-PCR Kit (TaKaRa, Tokyo, Japan).
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