Real-time PCR was used for quantification of pathogen colonization in planta using an ABI7300 PCR machine (Applied Biosystems) in combination with the qPCR Core kit for SYBR Green I (Eurogentec, Maastricht, The Netherlands) and analyzed using the 7300 System SDS software (Applied Biosystems). Unless described otherwise, the primer pair AtRub-F4 and AtRub-R4 targeting the gene encoding the large subunit of RuBisCo was used as endogenous control. Verticillium and R. solanacearum colonization was assessed as previously described [13] (link), [23] (link).
Abi 7300 pcr machine
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The ABI 7300 PCR machine is a real-time PCR system designed for sensitive and accurate nucleic acid detection and quantification. It features a 96-well block and can perform a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
7300 system sds software, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green qpcr master mix, by Tiangen Biotech (2 mentions)
Qpcr core kit for sybr green 1, by Eurogentec (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Primescript rt pcr kit, by Takara Bio (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Reverse transcriptase, by Promega (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
8 protocols using abi 7300 pcr machine
1
Real-time PCR for Pathogen Quantification
2
Gene Expression Analysis of Neuroinflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted by homogenization of the ipsilateral hemispheres in TRIzol reagent (Invitrogen, Camarillo, CA, USA) and was quantified with spectrophotometry. The RNA was reverse transcribed using RevertAid First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA). The PCR was performed using an ABI 7300 PCR machine (Applied Biosystems, Foster City, USA). Gene expression was normalized to β-actin. Primer sequences for CXCL1, CX3CL1, CCR1 and GAPDH were as follows: CXCL1 (forward, 5′-ACTGCACCCAAACCGAAGTC-3′, reverse, 5′-CAAGGGA GCTTCAGGGT-CAA-3′), CX3CL1 (forward, 5′-GCACAGGATGCAGGG CTTAC-3′, reverse, 5′-TGTCAGCCGCCTCAAA-ACT-3′), CCR1 (forward, 5′-CTGAGGGCCCGAA CTGTTAC-3′, reverse, 5′-GGCTAGGGCCCAGGTGAT-3′), GAPDH (forward, 5′-AATGT GTCCGTCGTGGATCTGA-3′, reverse, 5′-GATGCCTGCTTCACCACCTTCT-3′).
3
Quantitative Gene Expression Analysis of Botrytis cinerea
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First-strand cDNA was synthesized from 1 µg total RNA with Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Leiden, The Netherlands) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using an ABI7300 PCR machine (Applied Biosystems, Foster City, CA, USA) in combination with the qPCR SensiMix kit (Bioline, London, United Kingdom) using primers listed in Table S9 . qRT-PCR conditions were as follows: an initial 95°C denaturation step for 10 min, followed by denaturation for 15 s at 95°C and annealing/extension for 1 min at 60°C, for 40 cycles. The data were analyzed on the 7300 System SDS software (Applied Biosystems, Foster City, CA, USA). The gene expression values were normalized to the B. cinerea tubulin gene, BctubA (85 (link)).
4
Quantitative RT-PCR Analysis of EPC Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, total RNA was extracted from EPCs using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), and the reverse transcription reaction was performed with the synthesis kit (Bio‐Rad, Hercules, CA, USA), as per the manufacturer's instructions. Quantitative PCR (qPCR) was performed with the Prime Script RTPCR Kit (TaKaRa, Tokyo, Japan) using the ABI 7300 PCR machine (Applied Biosystems, Foster City, CA, USA). Amplification of each target mRNA was performed according to the following steps: denaturation for 30 seconds at 95°C, followed by 42 cycles, consisting of 5 seconds at 94°C and 32 seconds at 60°C. For each reaction, a melting curve was generated to test for primer dimer formation and false priming. The sequences of the forward and reverse primers are listed in Table 2 .
5
Quantifying miR-31 and RhoBTB1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from transfected A-431 cells using TRIzol reagent (Invitrogen, ThermoFisher Scientific, Inc.) and then reverse-transcribed into cDNA. RT-qPCR was performed using the SYBR Green qPCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China) on an ABI 7300 PCR machine (Applied Biosystems, Inc., Foster, CA, USA). The sequences of the primers used to detect miR-31 and U6 were as follows: miR-31, forward 5′-GGAGAGGCAAGATGCTGGCA-3′; U6, forward 5′-CGCAAGGATGACACGCAAATTC-3′; and a universal downstream reverse primer, 5′-GTGCAGGGTCCGAGGT-3′. The primers used for detection of RhoBTB1 were as follows: forward 5′-GGAGTGAAGGAGCCTGTGAG-3′; and reverse 5′-TGCCAATGAACCCCTTACTC-3′. qPCR cycling conditions were as follows: 95°C for 10 min, and then 95°C for 15 sec and 50°C for 2 min, for 40 cycles, followed by 60°C for 1 min. The melting curve was 65–95°C. The relative mRNA expression levels were calculated as 2−∆∆Cq and were normalized against U6.
6
Quantitative Expression Analysis of ABC Transporters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using the TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) chloroform extraction method. The cDNA was prepared from 1 µg total RNA using reverse transcriptase (Promega Corporation, Madison, WI, USA). Total cellular RNA was quantified by qPCR analysis using gene-specific primer pairs. RT-qPCR was performed by the SYBR-Green qPCR Master Mix (Tiangen Biotech Co., Ltd., Beijing, China) on an ABI 7300 PCR machine (Applied Biosystems, Inc., Foster, CA, USA). qPCR cycling conditions were as follows: 95°C for 5 min, and then 95°C for 15 sec, 65°C for 15 sec and 72°C for 32 sec, for 40 cycles. The melting curve was 60–95°C. The relative mRNA expression levels were calculated as 2−∆∆Cq method (18 (link)). Oligonucleotides used for the analysis were as follows: ABCB1 forward, 5′-CTAGAAGGTTCTGGGAAGAT and reverse, 5′-GAGTTTCTGTATGGTACCTG; ABCC1 forward, 5′-GGCTCAAGGAGTATTCAGAG and reverse, 5′-CCATCGATGATGATCTCTCC; EIF4G forward, 5′-CTCTGAGACCGTTGGGCAAA and reverse, 5′-GCGACGAATGCACCAATGT; ABCG2 forward, 5′-GCAGGTCAGAGTGTGGTTTC and reverse, 5′-GACAGCCAAGATGCAATGGT; and 18S ribosomal RNA forward, 5′-CCTGGATACCGCAGCTAGGA and reverse, 5′-GCGGCGCAATACGAATGCCCC. Each reaction was performed in triplicate and three independent experiments were run.
7
Isolation and Quantification of Gene Expression in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA was isolated from the cells using purification mini kits (RNeasy; Qiagen, Hilden, Germany) according to the manufacturer’s specifications. One microgram of total RNA was converted to cDNA (iScript cDNA Synthesis kit; Bio-Rad, Hercules, CA, USA) and was subsequently subjected to RT-PCR. PCR was performed in a thermal cycler at 94°C for 5 min followed by 30 amplification cycles (94°C, 30 sec; 55°C, 30 sec; 72°C, 1 min). Quantitative PCR was carried out to quantify the expression of CLU, Nestin, microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and β-actin messenger RNA on an ABI 7300 PCR machine (Applied Biosystems, Foster City, CA, USA). The relative expression value of the target gene was calculated as the ratio of target cDNA to β-actin. The primers used in this study are presented in Table I .
8
Quantifying Type I IFN Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IFN scores to describe type I IFN activation in our patients were analyzed as described elsewhere (26 (link)). In brief, total RNA was isolated from peripheral blood mononuclear cells (PBMCs) separated by Ficoll density gradient centrifugation with the RNeasy Mini Kit (Qiagen, Hilden, Germany) followed by DNase I digestion. Real-Time qRT-PCR was conducted with the Taqman Universal PCR Master Mix on an ABI 7300 PCR machine (Applied Biosystems, Darmstadt, Germany). IFN scores were calculated from the median fold change in relative mRNA expression of seven IFN-stimulated genes (IFI27, IFI44, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) normalized to HPRT and GAPDH as previously described (27 (link)). The following threshold were applied: <12.49: no increase in IFN signature; 12.49-30, weak IFN activation; 30-60, moderate IFN activation; 60-200, strong IFN activation; >200, very strong IFN activation (27 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!