To induce ER stress, N2a cells were treated with Thapsigargin (100 nM for 6 hours) as a positive control. Total RNA was isolated using TRIzol reagent and the PureLink Micro kit (Invitrogen, #12183016) followed by conversion to complementary DNA using the iScript system (Biorad #1708840). Quantitative RT-PCR was performed with iQ SYBR green supermix reagent (Biorad #1708880). Primers for transcripts reflecting ER stress induction were previously published (Oslowski and Urano, 2011 (link)), and β-actin was used as the control.
Purelink micro kit
Manufactured by Thermo Fisher Scientific
The Purelink Micro Kit is a product designed for the rapid and efficient extraction of high-quality DNA from small sample sizes. It utilizes a simple spin-column format to isolate DNA from a variety of sample types, including cells, tissues, and body fluids.
Automatically generated - may contain errors
Lab products found in correlation
Pet membranes, by Leica (2 mentions)
Lmd6500 laser capture microdissection imaging unit, by Leica (2 mentions)
Superscript 2 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Iq sybr green supermix reagent, by Bio-Rad (1 mentions)
Iscript system, by Bio-Rad (1 mentions)
Nanodrop nd 100 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using purelink micro kit
1
Thapsigargin-Induced ER Stress in N2a Cells
2
Sequencing and Analysis of Antibiotic Resistance Genes
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Sequencing of the PCR products from the blaTEM and blaKPC genes was performed using the Applied Biosystems/Hitachi automated sequencer, after purification of the PCR amplicons using the PureLink Micro Kit (Invitrogen), in accordance with the manufacturer's instructions. The blaTEM gene was sequenced in the K3C and K16R isolates because of phenotypic resistance that was presented in relation to the third-generation cephalosporins that were tested. Only the blaKPC gene was sequenced in K652 isolate, due to phenotypic resistance to carbapenems that was presented. The primers were the same as for PCR, plus TEMup21: 5′-TCCCTTTTTTGCGGCATTTTGC-3′ and TEMdwn280: 5′-CAGTGAGGCACCTATCTC-3′ [22 (link)], for the blaTEM gene, and KPC F: 5′-GAGCTGAACTCCGCCATC-3′ and KPC R: 5′-TATTTTTCCGAGATGGGTGAC-3′ [23 (link)], for the blaKPC gene.
The analyses on the DNA sequence and the multiple alignments were performed using the DNAstar software and BLAST [24 ]. The sequences for the blaTEM-1, blaTEM-15, and blaKPC-2 genes were deposited in the GenBank database under the access numbers KF906436, KF906435, and KF906437, respectively.
The analyses on the DNA sequence and the multiple alignments were performed using the DNAstar software and BLAST [24 ]. The sequences for the blaTEM-1, blaTEM-15, and blaKPC-2 genes were deposited in the GenBank database under the access numbers KF906436, KF906435, and KF906437, respectively.
3
Mite RNA Extraction with Trizol
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Mite family samples were individually crushed for 30 s in 500 μL of trizol and 5 μL of carrier RNA (5 ng/μL – Invitrogen). Samples were incubated for 5 min at RT and after transfer into 1.5 mL Eppendorfs, 100 μL of chloroform was added. Samples were hand-shaken for 15 s and let set for 3 min at RT. They were then centrifuged at 12,000 rcf for 15 min at 4 °C. The upper phase was gently pipetted out. Total RNA was extracted following the Purelink Micro kit (Invitrogen), eluted in 10 μL nuclease-free water and stored at −80 °C until further processing. Within each batch of 20–30 samples, one “blank” extraction was performed using only trizol, to test for contamination. RNA yield, concentration and quality were measured using a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies).
4
Laser Capture Microdissection of Astrocytes and Neurons
5
Laser Capture Microdissection of Astrocytes and Neurons
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Dissected brains were immediately frozen in liquid nitrogen, embedded in optimal cutting temperature compound (OCT; Tissue Tek) and 12 μm cryosections were acetone-fixed on PET membranes (Leica). Slides were dried, rinsed, stained with Cresyl Violet, washed and laser dissection was performed on an LMD6500-Laser Capture Microdissection/Imaging Unit (Leica). Regions enriched for astrocytes and neurons were dissected and used for RNA isolation with the Purelink Micro Kit (Invitrogen) according to the manufacturer’s protocol.
6
Quantitative Analysis of CCSER1 in Zebrafish Brains
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2-month old zebrafish brains dissected in PBS pH 7.0 were used for RNA extraction. 6 zebrafish per group (wild-type, CCSER1 P47F, and CCSER1 S48Y*) were analyzed. Tissue was homogenized in lysis buffer consisting of Trizol and RNA carrier. RNA was extracted with PureLink Micro Kit (ThermoFisher) based on the manufacturer's protocol. The concentration and purity of the RNA was determined using NanoDrop 2000 Spectrophotometer (ThermoFisher). Each brain sample yielded approximately 100 ng/μL of RNA. Quantitative real-time -PCR (qRT-PCR)
The relative expression of the CCSER1 was examined by qRT-PCR. cDNA was prepared by SuperScript II First-strand Synthesis System (Invitrogen). The cDNA was diluted with nuclease-free water to 100 ng/μL. The qRT-PCR amplification mixture (20 μL) contained 100 ng of cDNA, 10 μL 2X Go TaqqPCR Master Mix (Promega), and 300 nM forward and reverse primer. Reactions were carried out in triplicates using 7500 Real-time PCR System (Applied Biosystems) in a 96-well plate. The data were averaged and normalized to β-actin and Elif to obtain the ΔCT values (geomean). All PCR efficiencies were above 95%. Sequence Detection Software (version 1.3; Applied Biosystems) results were exported for calculations.
The relative expression of the CCSER1 was examined by qRT-PCR. cDNA was prepared by SuperScript II First-strand Synthesis System (Invitrogen). The cDNA was diluted with nuclease-free water to 100 ng/μL. The qRT-PCR amplification mixture (20 μL) contained 100 ng of cDNA, 10 μL 2X Go TaqqPCR Master Mix (Promega), and 300 nM forward and reverse primer. Reactions were carried out in triplicates using 7500 Real-time PCR System (Applied Biosystems) in a 96-well plate. The data were averaged and normalized to β-actin and Elif to obtain the ΔCT values (geomean). All PCR efficiencies were above 95%. Sequence Detection Software (version 1.3; Applied Biosystems) results were exported for calculations.
7
Quantitative Analysis of CCSER1 in Zebrafish Brains
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2-month old zebrafish brains dissected in PBS pH 7.0 were used for RNA extraction. 6 zebrafish per group (wild-type, CCSER1 P47F, and CCSER1 S48Y*) were analyzed. Tissue was homogenized in lysis buffer consisting of Trizol and RNA carrier. RNA was extracted with PureLink Micro Kit (ThermoFisher) based on the manufacturer's protocol. The concentration and purity of the RNA was determined using NanoDrop 2000 Spectrophotometer (ThermoFisher). Each brain sample yielded approximately 100 ng/μL of RNA. Quantitative real-time -PCR (qRT-PCR)
The relative expression of the CCSER1 was examined by qRT-PCR. cDNA was prepared by SuperScript II First-strand Synthesis System (Invitrogen). The cDNA was diluted with nuclease-free water to 100 ng/μL. The qRT-PCR amplification mixture (20 μL) contained 100 ng of cDNA, 10 μL 2X Go TaqqPCR Master Mix (Promega), and 300 nM forward and reverse primer. Reactions were carried out in triplicates using 7500 Real-time PCR System (Applied Biosystems) in a 96-well plate. The data were averaged and normalized to β-actin and Elif to obtain the ΔCT values (geomean). All PCR efficiencies were above 95%. Sequence Detection Software (version 1.3; Applied Biosystems) results were exported for calculations.
The relative expression of the CCSER1 was examined by qRT-PCR. cDNA was prepared by SuperScript II First-strand Synthesis System (Invitrogen). The cDNA was diluted with nuclease-free water to 100 ng/μL. The qRT-PCR amplification mixture (20 μL) contained 100 ng of cDNA, 10 μL 2X Go TaqqPCR Master Mix (Promega), and 300 nM forward and reverse primer. Reactions were carried out in triplicates using 7500 Real-time PCR System (Applied Biosystems) in a 96-well plate. The data were averaged and normalized to β-actin and Elif to obtain the ΔCT values (geomean). All PCR efficiencies were above 95%. Sequence Detection Software (version 1.3; Applied Biosystems) results were exported for calculations.
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