SSH was performed with the PCR-SelectTM cDNA subtraction kit (Clontech) according to the manufacture's protocol. In brief, 2.0 μg of poly A+ mRNA, each from the pcDNA3.1 (-)/myc-His A-HBVDNAPTP1 driver group and the pcDNA3.1 (-)/myc-His A tester group was subjected to cDNA synthesis, respectively. After restriction with RsaI, small sizes of cDNAs were obtained. The tester cDNAs were then subdivided into two parts, ligated with the specific adaptor 1 and adaptor 2, respectively. After two subtractive hybridization reactions and two suppression PCR amplifications, differentially expressed cDNAs were selectively amplified. Then the second PCR products were used as templates for PCR amplification of G3PDH (a housekeeping gene) at 18, 23, 28, 33 cycles, respectively, to analyze subtraction efficiency. The second PCR products were directly purified using Wizard PCR-Preps DNA Purification System (Promega), and inserted into pGEM-T Easy (Promega) to construct the subtracted library. Colony PCRs were conducted to confirm the size of cDNA inserts being ranged between 200 and 1000 bp by using T7/SP6 specific primers localized in pGEM-T Easy. After DNA sequencing of the positive colonies, nucleotide homology searches were performed using the BLAST program at NCBI.
Pcr selecttm cdna subtraction kit
Manufactured by Takara Bio
Sourced in United States
The PCR-SelectTM cDNA subtraction kit is a laboratory tool designed to identify differentially expressed genes between two samples. It utilizes a suppression PCR-based technique to selectively amplify unique sequences from one sample relative to another.
Automatically generated - may contain errors
Lab products found in correlation
Wizard pcr preps dna purification system, by Promega (1 mentions)
Pgem t easy, by Promega (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130 analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Oligotex mrna spin column kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
Smartertm pcr cdna synthesis kit, by Takara Bio (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Nanodroptm 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Escherichia coli dh5α, by Thermo Fisher Scientific (1 mentions)
6 protocols using pcr selecttm cdna subtraction kit
1
SSH Subtraction Library Construction
2
Subtractive Hybridization for Differential Gene Expression
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For subtractive hybridization, 1.0 μg of each total RNA mixture was employed to produce double-stranded cDNA using the Super SMARTTM cDNA Synthesis Kit (Clontech Laboratories Inc., Palo Alto, CA, USA) according to the manufacturer’s instructions. To determine which genes were differentially expressed during the interaction with S. sclerotiorum, a forward subtractive cDNA library was constructed using the PCR-SelectTM cDNA Subtraction Kit (Clontech Laboratories Inc., Palo Alto, CA, USA) according to the standard protocol provided. The subtracted cDNA population was cloned into a PCR 4®-TOPO vector (Invitrogen Corp., Carlsbad, CA, USA) and used to transform One Shot® Top 10 EletrocompTMEscherichia coli cells (Invitrogen Corp., Carlsbad, CA, USA). The plasmid DNA was obtained using alkaline lysis. The cloned products were sequenced using a M13 forward primer and the Big Dye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA). The automated capillary electrophoresis sequencing runs were performed on an ABI prism 3130 analyzer (Applied Biosystems, Carlsbad, CA, USA).
3
RNA Extraction and cDNA Synthesis
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Total RNA from time 0 h samples and time 24 h-shreds was isolated following the hot borate method29 (link). Isolation of poly A+ mRNA from total RNA was performed using the Oligotex mRNA Spin-Column kit (Qiagen). Purified poly A+ mRNA from time 0 h samples and time 24 h-shreds were used for cDNA synthesis by the PCR-SelectTM cDNA subtraction kit (Clontech).
4
Transcriptome Analysis of Plant Infection Response
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Total RNA was extracted from harvested leaves using the TriPure Isolation Reagent (Roche Applied Sciences, Basel, Switzerland) according to the manufacturer's instructions. It was then quantified using a Thermo Scientific NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Total RNA was assessed by formamide denaturing gel electrophoresis, and mRNA isolated from the total RNA using Dynabeads Oligo (dT)25 isolation beads (Invitrogen, Thermo Fisher Scientific, Whaltman, MA, USA). An SSH cDNA library was constructed using the PCR-SelectTM cDNA Subtraction Kit (Clontech, Mountain View, CA, USA). Equal amounts of total RNA sampled from inoculated plants were pooled. Two micrograms of this mRNA pool was used for cDNA synthesis. cDNAs were also derived from mock-inoculated leaves harvested at the same time points. Subtractive hybridization was performed using sample cDNA (tester) from the inoculated plants, which was subtracted with cDNA (driver) from the non-inoculated plants. This forward subtraction identifies genes induced (upregulated) during the infection process.
5
Transcriptomic Analysis of Fungal Resistance
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Total RNA was extracted from all the samples with TRI reagent (Sigma-Aldrich, USA). The quality of RNA was checked in an agarose gel and quanti ed in a NanoDrop TM 1000 spectrophotometer (Thermo Scienti c, USA). 1µg of puri ed RNA was used for cDNA synthesis following the manufacturer's instructions of Smarter TM PCR cDNA synthesis kit (Clontech, CA, USA). Forward and reverse subtractions for the cDNAs were done following the manufacturer's instructions of PCR-Select TM cDNA subtraction kit (Clontech, CA, USA). Forward subtraction represents resistance response library (RRL), in which cv Co 7805 challenged with pathotype Cf87012 was used as tester, cv Co 7805 challenged with Cf94012 and mock sample of cv Co 7805 were used as the driver. In reverse subtraction representing susceptible response library (SRL), the cv Co 7805 challenged with Cf94012 was used as tester and cv Co 7805 challenged with the pathotype Cf87012 and mock sample of cv Co 7805 were used as driver.
6
Subtraction Library Construction Protocol
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SSH libraries were constructed using a PCR-Select TM cDNA Subtraction kit (Clontech, USA) based on the manufacturer instructions. The cDNAs from the green and albino leaves were mutually used as testers and drivers, and they were forward and reverse subtracted. Subsequently, these subtracted cDNAs were cut by RsaI, mixed for 1.5 h at 37°C, and then ligated to adaptor 1 (5'-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG GCA GGT-3') and adaptor 2 (5'-ACC TCG GCC GCG ACC ACG CTG CCC TAT AGT GAG TCG TAT TAG-3'). After two rounds of subtractive hybridization, two rounds of suppression PCR amplification were successively carried out. The efficiency of suppression subtractive hybridization was determined by gel electrophoresis. The purified PCR products were transformed into Escherichia coli DH5 α (Invitrogen, USA), and the recombinant clones were plated onto LB medium containing ampicillin, X-Gal, and IPTG, and were incubated overnight at 37°C. All of the white clones were picked for further analyses and sequencing.
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