RNA was isolated from cultured ZEST cells using a QIAGEN RNeasy kit, and cDNA was generated using a BioRad iScript cDNA synthesis kit. qRT-PCR was performed on an Eppendorf Mastercycler with BioRad SsoAdvanced Universal SYBR Green Supermix. Primers have been previously described36 (link),37 (link). Fold expression was determined by the ΔΔCt method, using ef1α as a reference gene, and kidney as a reference tissue.
Mastercycler
Manufactured by Bio-Rad
Sourced in United States
The Mastercycler is a thermal cycler designed for performing polymerase chain reaction (PCR) experiments. It provides precise temperature control and programmable cycling for a variety of PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Icycler machine, by Bio-Rad (3 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Sybr green reagent, by Bio-Rad (2 mentions)
Iscript reverse transcription supermix kit, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rneasy kit, by Bio-Rad (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Random hexamer primer, by Thermo Fisher Scientific (1 mentions)
Oligo dt 18 primer, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
10 protocols using mastercycler
1
qRT-PCR Analysis of ZEST Cell Gene Expression
2
Isolation and Characterization of CsTCTP Genes
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The nucleotide sequence of Cucumis sativus L. TCTPs (CsTCTP1, accession: XP-004134215 and CsTCTP2, accession: XP-004135602) was retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/ ). Amplifications of CsTCTP1 and CsTCTP2 cDNAs were performed with primers 1- cDNA F/1- cDNA R and 2- cDNA F/2- cDNA R on first-strand cDNA templates of B21-a-2-1-2 and B21-a-2-2-2 in a Mastercycler (BIO-RAD) under the following conditions: 94°C for 5 min, 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, 32 cycles, and 72°C for 10 min. The PCR products were gel-purified, cloned into pMD18-T vector (TaKaRa, China) and sequenced (Sangon Biotech, China).
Genomic PCRs were performed with primers 1- DNA F/1- DNA R and 2- DNA F/2- DNA R on DNA templates of B21-a-2-1-2 and B21-a-2-2-2 as follows: 94°C for 5 min, 94°C for 30 s, 62°C for 30 s, 72°C for 2 min, 36 cycles, and 72°C for 10 min. For promoter analysis, the promoter regions of CsTCTP1 and CsTCTP2 (approximately -2.0 kb upsteam of translation initiation site) were also isolated from B21-a-2-1-2 and B21-a-2-2-2 genomic DNA based on 1- P F/1- P R primers and 2- P F/2- P R primers. PCR conditions used were as follows: 94°C for 5 min, 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, 38 cycles, and 72°C for 10 min. The PCR products were gel purified, cloned and sequenced.
Genomic PCRs were performed with primers 1- DNA F/1- DNA R and 2- DNA F/2- DNA R on DNA templates of B21-a-2-1-2 and B21-a-2-2-2 as follows: 94°C for 5 min, 94°C for 30 s, 62°C for 30 s, 72°C for 2 min, 36 cycles, and 72°C for 10 min. For promoter analysis, the promoter regions of CsTCTP1 and CsTCTP2 (approximately -2.0 kb upsteam of translation initiation site) were also isolated from B21-a-2-1-2 and B21-a-2-2-2 genomic DNA based on 1- P F/1- P R primers and 2- P F/2- P R primers. PCR conditions used were as follows: 94°C for 5 min, 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, 38 cycles, and 72°C for 10 min. The PCR products were gel purified, cloned and sequenced.
3
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using the RNAiso Plus reagent(Takara, Dalian, China), and then genomic DNA Contaminated gDNA was removed as follows: 2 μL (1 μg) total RNA, 1 μL gDNA Eraser, 2 μL 5× gDNA Eraser Buffer, 5 μL RNase free water were mixed in PCR tube, the reaction was incubated at 42°C for 2 min. Afterwards, 1 μL PrimeScript RT Enzyme Mix I, 1 μL RT Primer Mix, 4 μL 5× PrimeScript Buffer 2, 4 μL RNase free water were added to the tube. The mixture was incubated at 37°C for 15 min, 85°C for 5 s. The cDNA was stored at −20°C for later use. was removed and reverse-transcribed in a BIO-RAD Mastercycler (BIO-RAD, California, USA), using a PrimeScript™ RT reagent Kit (Takara, Dalian, China) with gDNA Eraser according to the manufacturers’ instructions. PCR was performed in triplicate in a LightCycler®96 system(Roche Diagnostics GmbH, Mannheim, Germany). The sequences of the specific primers are shown in Table 1
4
Gene Expression Analysis in Murine Macrophages
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Total RNA was extracted from mouse bone-marrow-derived macrophages and RAW264.7 Dox-Cas9 cells harboring non-targeting ROSA or Huwe1-targeting sgRNAs. 0.5×106 cells were lysed using Trizol reagent (Thermo-Fisher Scientific, 5596–018) and total RNA was isolated as recommended, and treated with 0.2 U/μl Turbo DNase (Thermo Fisher Scientific, AM2238). cDNA was prepared using Oligo (dT18) Primer (Thermo Fisher Scientific, S0132) or random hexamer primers (Thermo Fisher Scientific, S0142) and RevertAid Reverse Transcriptase (Thermo-Fisher Scientific, EP0441). Real-time PCR experiments were run on a Mastercycler (Biorad), using SYBR Green (Thermo-Fisher Scientific, S7567). Primers for qPCR are listed in Table 6 .
5
RNA Extraction and qPCR from Hydrogel Cell Culture
6
Zebrafish RNA Extraction and qPCR Analysis
7
mRNA Expression Analysis in ZR751 Cells
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The ZR751 cells (2 × 105 cells/well) were cultured in 6-well culture plates, treated with or without test samples for 24 h. After incubation, total RNA was prepared using an RNeasy kit (Qiagen) and primed with random hexamers to synthesize complementary DNA using superscript II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was carried out in a Mastercycler (Biorad) with specific primers (Additional file
1 : Table S1). Amplification products obtained by PCR were electrophoretically separated on 1.5% agarose gel, stained with ethidium bromide (0.5 μg/ml) and visualized under gel documentation system (Bio-Rad).
8
Hydrogel RNA Isolation and qPCR Analysis
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Total RNA was isolated from hydrogels using TRI Reagent (Sigma-Aldrich). In brief, gels were digested using 2 mg/ml Collagenase Type I (Worthington Biochemical Corporation) to collect cells that were then lysed in 500 μl of TRI Reagent. RNA was isolated using a chloroform extraction and excess 2-propanol (Sigma-Aldrich) to precipitate it, and pellets were washed and resuspended in RNAse free water (Sigma-Aldrich). The RNA concentration and quality were assessed with a ND-1000 Nanodrop Spectrophotometer. For RT-qPCR, cDNA was synthesized with the iScript Reverse Transcription Supermix kit (Bio-Rad) and an Eppendorf Mastercycler. Relative mRNA expression levels were measured using SYBR Green reagents (Bio-Rad) using an iCycler machine (Bio-Rad), normalizing to the housekeeping gene (ribosomal protein L30). Custom primers are presented in Table 1 in the supplemental material.
9
Evaluating Transcriptomic Changes in Gene-Edited Cells
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Total RNA was extracted from RKO-Dox-Cas9-P2A-GFP cells harboring either AAVS1/CCR5, AAVS1/IRF1, SPOP/CCR5, SPOP/IRF1-targeting sgRNAs. 1×106 cells were lysed using Trizol reagent (Thermo Fisher Scientific, 5596-018) and treated with Turbo DNase (Thermo Fisher Scientific, 10792877). cDNA was prepared using random hexamer primer and RevertAid Reverse Transcriptase (Thermo Fisher Scientific, EP0441). Real-time PCR experiments were run on a Mastercycler (Bio-Rad), using SYBR Green (Thermo Fisher Scientific). Primers for qPCR are listed in Supplementary file 6 .
10
Quantitative Real-Time PCR
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