Total RNA was isolated from IMR-32 and SH-SY5Y cells using the GeneJet RNA Purification Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific) and qPCR performed using Maxima qPCR Master Mix. Sequences of primers were as follows: ALK forward primer: 5′-GTGCCATGCTGCCAGTTAAG-3′, ALK reverse primer: 5′-TGGTTGCTTTTGCTGGGGTA-3′; MDK forward primer: 5′-AAGGAGTTTGGAGCCGACTG-3, MDK reverse primer: 5-CATTGTAGCGCGCCTTCTTC-3′. Amplification of GAPDH was used as a normalization control for total RNA input. Relative expression of ALK and MDK were determined using the dCq method.
Maxima first strand cdna synthesis kit for rt pcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Maxima First Strand cDNA Synthesis Kit for RT-PCR is a reverse transcription kit designed for the synthesis of first-strand cDNA from total RNA or poly(A)+ RNA. The kit includes optimized reagents and protocols for efficient and reliable cDNA synthesis.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Avantor (5 mentions)
Peqgold, by Avantor (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Genejet rna isolation kit, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3 pcr detection system, by Thermo Fisher Scientific (3 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Lightcycler 480, by Roche (2 mentions)
Sybr green fluorescent mix, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Mysticq microrna cdna synthesis mix, by Merck Group (2 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Maxima qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Ariamx real time pcr system, by Agilent Technologies (1 mentions)
Sybr green 1 master mix, by Roche (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Light cycler faststart dna master sybergreen 1 kit, by Roche (1 mentions)
Transcriptor first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Quantstudio 7 real time pcr system, by Thermo Fisher Scientific (1 mentions)
16 protocols using maxima first strand cdna synthesis kit for rt pcr
1
Quantification of ALK and MDK Expression
2
Evaluating B. abortus Virulence Genes
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The relative expression of several genes codifying for T4SS virB, its effector proteins and transcription factors involved in the virulence and intracellular survival of B. abortus Δ270 was evaluated in infected macrophages by the 2−ΔΔCT method. For this, 1 × 106 RAW264.7 macrophages/well were infected at MOI 1:10 for 24 h with the strains wt, mutant and inactivated. Then, total RNA was extracted with TRIzol (Thermo Fisher Scientific Inc., MA, United States) as was indicated by the manufacturer. Complementary DNA (cDNA) was obtained from RNA by reverse transcription using the Maxima First Strand cDNA Synthesis kit for RT-PCR (Thermo Fisher Scientific Inc., MA, United States) and the relative expression of the genes of interest (Table 3 ) was quantified using the Takyon q-PCR kit for SYBR assays by means of the AriaMx Real Time PCR system (Agilent Technologies, CA, United States). gyrA and 16s housekeeping genes were used as reference genes for all assays. All assays were done in triplicate.
3
Quantitative Gene Expression Analysis
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1x105 cells were seeded per well in a 6-well plate and cultured with an IC50 concentration of the examined compounds for 48 h. Total RNA from the cells was extracted using TRI Reagent (Sigma, Germany) according to the manufacturer’s instructions. The RNA concentrations were quantified using an UV spectrophotometer (NanoDrop ND-1000 Spectrophotometer, Thermo Scientific, USA). Then cDNA was synthesized from equal amounts of RNA using a Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific, USA). The primer sequences used were as follows: ERK1 GenBank: ABCB1 forward, 5′-TGGAAACAGTGGCTGTGGGAAG-3′, and reverse, 5′-TCCTGTCCATCAACACTGACCATC-3′; HMBS GenBank: NM_002745 forward, 5′-GGCAATGCGGCTGCAA-3′, and reverse, 5′-GGGTACCCACGCGAATCAC-3′; HPRT1 GenBank: NM_001101 forward, 5′-TGACACTGGCAAAACAATGCA -3′, and reverse, 5′-GGTCCTTTTCACCAGCAAGCT-3′. The expression levels of the different genes were measured by real-time PCR amplification in conjunction with a SYBR Green I Master Mix (Roche) using a LightCycler 480 (Roche, Basel, Switzerland). Finally, threshold cycle numbers (Ct) were used to calculate the expression of each target gene by normalizing to the house-keeping genes hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).
4
Quantitative Real-Time PCR Analysis of Apoptosis Regulators
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HCT-116 cells were treated as described in Section 2.9 . Total RNA was isolated from the cells using Rneasy Plus Mini Kit (QIAGEN) according to the manufacturer’s instructions, and immediately frozen at −80 °C until further use. DNase-treated RNAs were used to synthesize cDNA with the Transcriptor First Strand cDNA Synthesis Kit, using random hexamers as specified by the manufacturer (Maxima First Strand cDNA Synthesis Kit for RT-PCR by Thermo).
Real-time PCR amplification (RT-PCR) and advanced relative quantification analysis were achieved using a Light Cycler 480 instrument (Roche Applied Science, Penzberg, Germany) with software version LCS480 1.5.039. All reactions were performed in duplicate with the Light Cycler Fast Start DNA Master SYBER Green I Kit (Roche Applied Science) in a final 20 µL volume with 2.5 mM MgCl2, 0.2 µM of each primer and 2 µL cDNA. Amplification conditions consisted of an initial pre-incubation step at 95 °C for 10 min (polymerase activation), followed by amplification of the target cDNA for 45 cycles (95 °C for 15 s, 60 °C for 20 s, and extension time at 72 °C for 30 s) [35 (link)]. the primer sequences (5′ to 3′) for qPCR amplification were:
Real-time PCR amplification (RT-PCR) and advanced relative quantification analysis were achieved using a Light Cycler 480 instrument (Roche Applied Science, Penzberg, Germany) with software version LCS480 1.5.039. All reactions were performed in duplicate with the Light Cycler Fast Start DNA Master SYBER Green I Kit (Roche Applied Science) in a final 20 µL volume with 2.5 mM MgCl2, 0.2 µM of each primer and 2 µL cDNA. Amplification conditions consisted of an initial pre-incubation step at 95 °C for 10 min (polymerase activation), followed by amplification of the target cDNA for 45 cycles (95 °C for 15 s, 60 °C for 20 s, and extension time at 72 °C for 30 s) [35 (link)]. the primer sequences (5′ to 3′) for qPCR amplification were:
Apaf-1 for-AACCAGGATGGGTCACCATA
Apaf-1 rev-ACTGAAACCCAATGCACTCC
NOXA for-CAGAGCTGGAAGTCGAGTG
NOXA rev-CAGGTTCCTGAGCAGAAGAG
5
Quantifying mRNA Levels via RT-qPCR
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Total RNA was extracted from cell samples using the miRNeasy mini kit (Qiagen). Complementary DNA (cDNA) was synthesized from 1,000 ng of RNA using the Maxima First Strand cDNA Synthesis Kit for RT–PCR (Thermo Fisher). qPCR was performed using standard SYBR green reagents and protocols on a QuantStudio 7 Real-Time PCR system (Applied Biosystems). All reactions were performed in technical triplicates. The expression of the target mRNA was determined using the ΔΔCt method using beta-Actin expression as a reference for each sample. All the primers utilized were designed using Primer 3 (http://frodo.wi.mit.edu/primer3/ ) and synthesized by Integrated DNA Technologies. Primer sequences are listed in SI Appendix, Table S2 .
6
Quantification of Hypoxia-Induced Gene Expression
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RNA was isolated using peqGold (Peqlab, Erlangen, Germany) and measured using a Nanodrop ND-1000 spectrophotometer (Peqlab). Reverse transcription was performed with the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Fisher Scientific, Waltham, USA). RNA expression of TMEM126B (fwd: 5‘-GGTGGTGTTCGGGTATGAGG-3‘, rev: 5‘-TCTTGAAAACCTTGGGCGCT-3‘), Glut1 (fwd: 5‘-TCACTGTGCTCCTGGTTCTG-3‘, rev: 5‘-CCTGTGCTCCTGAGAGATCC-3‘), IL-1β (fwd: 5‘-TCTTTAACGCAGGACAG-3‘, rev: 5‘-TTCGACACATGGGATAACGA-3‘), and HIF-1α (fwd: 5‘-GCTGGCCCCAGCCGCTGGAG-3‘, rev: 5‘-GAGTGCAGGGTCAGCACTAC-3‘) was analyzed using SYBR green fluorescent mix (Thermo Fischer Scientific) on a CFX96 Real Time PCR Detection System (Bio-Rad) and normalized to TBP (fwd: 5‘-GGGCCGCCGGCTGTTTAACT-3‘, rev:5‘-GGGCCGCCGGCTGTTTAACT-3‘). BNIP3 primers were purchased from Quiagen (Hilden, Germany).
7
Quantitative analysis of HUNK gene
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RNA was prepared by using the GeneJet RNA isolation kit (Thermo Scientific). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific). The resulting cDNA was used to perform quantitative RealTime PCR (QRT-PCR) using the Bio-Rad myIQ system. PrimePCR SYBR Green Assay for human HUNK was purchased from Bio-Rad. Primers for GAPDH are Forward-TGCACCACCAACTGCTTAGC and Reverse-GGCATGGACTGTGGTCATGAG.
8
Quantitative Real-Time PCR Analysis
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Total RNA from cells was extracted using AxyPrep Multisourse Total RNA Miniprep Kit (Axygen, Tewksbury, MA, USA) according to the manufacturer's protocol. First-strand cDNA was synthesised from total RNA using Maxima First Strand cDNA Synthesis Kit for RT–PCR (Thermo Scientific). Gene expression levels were measured by 7500 Fast Real-Time PCR System (Applied Biosystems, Beverly, MA, USA) using the KAPA SYBR FAST qPCR Kits (Kapa Biosystems, Woburn, MA, USA). Expression of each gene was normalised to β-actin as a reference. The forward and reverse primer sequences have been previously described (Leong et al, 2007 (link)). The conditions for all quantitative RT–PCR reactions are as follows: 2 min at 95 °C followed by 15 s at 95 °C and 30 s at 60 °C for 40 cycles. All PCR products were confirmed by the presence of a single peak upon melting curve analysis and by gel electrophoresis. Primers used for all quantitative RT–PCR were listed on Table 1 .
9
Gene Expression Analysis of Hunk in Mice
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RNA was isolated using the GeneJet RNA isolation kit (Thermo Scientific). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Scientific). RealTime PCR using PrimePCR mouse Hunk (Bio-Rad) was performed using the Bio-Rad myIQ. Hunk mRNA levels were normalized to Gapdh levels. Primers for Gapdh are: Forward-GCACAGTCAAGGCCGAGAAT, Reverse-GCCTTCTCCATGGTGGTGAA
10
RNA Expression Analysis by qRT-PCR
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RNA was isolated using peqGold (Peqlab, Erlangen, Germany) and measured by using a Nanodrop ND-1000 spectrophotometer (Peqlab, Erlangen, Germany). Reverse transcription was performed with the Maxima First Strand cDNA Synthesis Kit for RT-PCR (Thermo Fisher Scientific, Waltham, USA). RNA expression was analyzed by using a SYBR green fluorescent mix (Thermo Fischer Scientific) on a CFX96 Real Time PCR Detection System (Bio-Rad), and normalized to TATA box binding protein (TBP). All primer sequences are listed in Table 1 .
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