The SST-REX method was performed as described3 (link)4 (link). Total RNA was purified from LNCaP-CR and LNCaP cells using Trizol (Invitrogen) and mRNA was isolated using Fast-Track 2.0 kits (Invitrogen). cDNA was synthesized using random hexamers with SuperScript Choice System (Invitrogen) according to the manufacturer’s instructions and inserted into BstXI sites of pMX-SST vector3 (link)4 (link) using BstXI adaptors (Invitrogen). The ligated vectors were electroporated into E. coli using an E-coli Pulser (Bio-Rad) at 1.8 kV. Then, the cDNA library was prepared by culturing the transfected E. coli. High-titer retroviruses from the above cDNA library were produced using the packaging cell line Plat-E (Cell Biolabs). Ba/F3 cells were infected with the retroviruses using Polybrene (Chemicon). Ba/F3 clones that grew in the absence of IL-3 were selected. Genomic DNA extracted from the IL-3-independent Ba/F3 clones was applied to PCR to recover the integrated cDNAs using the PCR primers, 5′-TAATACGACTCACTATAGGGCGCGCAGCTGTAAACGGTAG-3′ and 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3′. The PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing kits with 5′-ATTAACCCTCACTAAAGGGAGGGGGTGGACCATCCTCTA-3′ as a primer.
Superscript choice system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Netherlands
The SuperScript Choice System is a cDNA synthesis kit designed for the efficient and reliable generation of first-strand cDNA from various RNA sources. The system utilizes a proprietary reverse transcriptase enzyme and optimized reaction conditions to facilitate high-yield cDNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Lightcycler 480, by Roche (4 mentions)
Rnaeasy, by Qiagen (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Rna bee, by Tel-Test (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Rnalater solution, by Thermo Fisher Scientific (2 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Bioarray highyield rna transcript labeling kit, by Enzo Life Sciences (2 mentions)
Tripure isolation reagent, by Roche (2 mentions)
Polybrene, by Merck Group (1 mentions)
E coli pulser, by Bio-Rad (1 mentions)
Plat e, by Cell Biolabs (1 mentions)
Lab on a chip bioanalyzer 2100, by Agilent Technologies (1 mentions)
Rna transcript labeling kit, by Enzo Life Sciences (1 mentions)
Mouse 430 2.0 genechip, by Thermo Fisher Scientific (1 mentions)
Genearray scanner g2500a, by Thermo Fisher Scientific (1 mentions)
G3000 genearray scanner, by Thermo Fisher Scientific (1 mentions)
32 protocols using superscript choice system
1
Screening for Growth Factor-Independent Genes
2
Transcriptional Profiling of MSCV Variants
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Independently derived 70Z/3 lines infected with MSCV, or MSCV-miR-24-2 were generated previously[65 (link)]. Four MSCV lines and 2 MSCV-miR-24-2 lines were examined. Total RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA). RNA quality was evaluated using the Lab-on-a-Chip Bioanalyzer 2100 (Agilent, Palo Alto, CA). Biotinylated cRNA was prepared according to the standard Affymetrix protocol using the Superscript Choice System (Invitrogen) and the RNA transcript labeling kit (ENZO, Farmingdale, NY) for cRNA preparation. Following fragmentation 10ug of cRNA was hybridized to Affymetrix Mouse 430 2.0 Gene Chip. Microarray analysis was performed using the Affymetrix GeneArray Scanner G2500A. The Affymetrix Expression Console Software Version 1.4.0 was used to create summarized expression values (CHP-files) from expression array feature intensities (CEL-files). Raw data were normalized with robust multichip analysis (RMA) with Affymetrix transcriptome analysis console (TAC) software. DNA microarray and sample annotation data were deposited in GEO under the accession number GSE65874.
3
Transcriptome Analysis Using Affymetrix Exon Array
4
Isolation and Characterization of Bitiscetin
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Total RNA was extracted from the B. arietans venom gland (1 g) using TRIZOL (Invitrogen, Carlsbad, CA, USA), and mRNA was isolated from total RNA by a Poly(A)Purist mRNA purification kit (Ambion, Austin, TX, USA). cDNA was synthesized by the SuperScript Choice system (Invitrogen) using an oligo-dT primer. This cDNA was used for bitiscetin-specific PCR experiments. In addition, the cDNA was also cloned into Lambda ZAP II vector (Stratagene, La Jolla, CA, USA) to create a library, and the amplified library was used to find the open reading frame ends. For all procedures we followed the instructions provided by the kit supplier.
5
RNA Isolation and cDNA Synthesis
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Mice were euthanized by cervical dislocation and a piece of tissue was isolated and placed in cold RNA later solution (Thermo Fisher Scientific) for 48 h at 4°C. RNA was isolated (RNAeasy; QIAGEN), and cDNA synthesis (SuperScript choice system; Invitrogen) was performed according to the manufacturer’s instructions.
6
RNA Isolation, cDNA Synthesis, and cRNA Labeling
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We isolated total RNA using Trizol reagent (Invitrogen, Carlsbad, CA, USA)
and purified the RNeasy Total RNA Isolation Kit (Qiagen, Hilden, Germany).
The SuperScript Choice system (Invitrogen) was used to synthesize
double-stranded cDNA. Phase Lock Gels-phenol and chloroform extractions
(Eppendorf, Hamburg, Germany) were used to clean up the cDNA template. We
then generated biotin-labeled cRNA from this template using a BIOARRAY
HIGHYIELD RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY,
USA). In vitro transcription products were purified using
RNeasy spin columns (Qiagen) and were quantified by spectrophotometric
analysis. After the purification, the cRNA was fragmented using the standard
procedure by Affymetrix to obtain a distribution of RNA fragments sized from
approximately 35 to 200 bases. Fragmented RNA was checked with agarose gel
electrophoresis.
and purified the RNeasy Total RNA Isolation Kit (Qiagen, Hilden, Germany).
The SuperScript Choice system (Invitrogen) was used to synthesize
double-stranded cDNA. Phase Lock Gels-phenol and chloroform extractions
(Eppendorf, Hamburg, Germany) were used to clean up the cDNA template. We
then generated biotin-labeled cRNA from this template using a BIOARRAY
HIGHYIELD RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, NY,
USA). In vitro transcription products were purified using
RNeasy spin columns (Qiagen) and were quantified by spectrophotometric
analysis. After the purification, the cRNA was fragmented using the standard
procedure by Affymetrix to obtain a distribution of RNA fragments sized from
approximately 35 to 200 bases. Fragmented RNA was checked with agarose gel
electrophoresis.
7
RNA Isolation and qRT-PCR Analysis
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RNA isolation (RNAeasy, QIAGEN) and cDNA synthesis (Superscript choice system, Invitrogen) was performed according to the manufacturer’s instructions. Quantitative RT–PCR was carried out using the QuantiTect SYBR Green RT-PCR Kit (QIAGEN) and validated primers (QIAGEN) according to the manufacturer’s instructions. The PCR reactions and detection were performed on a light cycler (Roche) using GAPDH as internal controls for normalization purposes. The corresponding PCR primers were provided in S1 Table .
8
Expression Cloning of MLL/AF9 Leukemia cDNA
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Expression cloning was performed as previously reported [13 (link)] with slight modifications. cDNA library was generated from MLL/AF9 leukemia cells using Superscript Choice System (Invitrogen) and BstXI adaptor (Invitrogen). cDNA fragments ranging from 1.0 to 5.0 kb were selected by Chroma spin column (Clontech, Mountain View, CA, USA) and electrophoresis on an agarose gel and subcloned into pMX retrovirus vector (a kind gift from Toshio Kitamura, Tokyo University, Tokyo, Japan). Retrovirus carrying cDNA library derived from MLL/AF9 leukemia cells was produced by transient transduction to Plat-E cells, and then infected to YB2/0 cells [13 (link)]. YB2/0 cells reacted with R54 or B2 mAbs were FACS-sorted and expanded. After third round of FACS-sorting, insert DNA was amplified from cDNA derived from the enriched cells, and sequenced.
9
Pancreatic RNA Extraction and qRT-PCR
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Mice were euthanized by cervical dislocation and the pancreatic tissue was isolated and placed in cold RNA later solution (Thermo Fisher Scientific) for 48 h at 4°C. RNA was isolated (RNAeasy; QIAGEN), and cDNA synthesis (SuperScript choice system; Invitrogen) was performed according to the manufacturer’s instructions. Quantitative RT–PCR was performed using validated primers (QIAGEN) and the QuantiTect SYBR Green RT-PCR kit (QIAGEN) following the manufacturer’s instructions. PCR reactions and detection were performed on a Mastercycler ep realplex cycler using GAPDH as an internal control for normalization purposes.
10
Biotin-Labeled cRNA Hybridization to Affymetrix GeneChip
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The SuperScript Choice system (Invitrogen, Carlsbad, CA, USA) was used for transcription of total RNA into double-stranded cDNA. To assembly first-strand cDNA synthesis a T7-(dT24) oligonucleotide primer with a RNA polymerase promoter site was used. In vitro transcription followed after second-strand synthesis in the presence of biotin-11-cytidine triphosphate and biotin-16-uridine triphosphate (Enzo Diagnostics, New York, NY, USA). Next, biotin-labelled cRNA were fragmented (20 μg at 94°C for 35 minutes) and added to a hybridization solution to a final cRNA concentration (0.05 mg/mL). The solution was incubated for hybridization with an oligonucleotide array (AffymetrixGeneChip [Hu133A]), containing 22,283 probe sets for known genes or expressed sequence tags (ESTs). After staining with streptavidin-phycoerythrin a Gene Array scanner G2500A (Hewlett Packard, Palo Alto, CA, USA) was used for scanning of the hybridisation products according to the recommendations of the manufacturer.
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