Total DNA was extracted from approximately 50 mg of leaf material from 14 putative transgenic lines using the CTAB method described in [65 ].Total RNA was extracted from approximately 50 mg of leaf tissue, collected from infected and mock inoculated plants and flash frozen in liquid nitrogen. The leaf tissue was ground into a fine powder using the Qiagen TissueLyser II system (Qiagen) and total RNA was extracted using Qiazol lysis reagent (ThermoFisher Scientific), according to the manufacturer's instructions. Total RNA was treated with RiboLock (ThermoFisher Scientific) and stored at −80 °C.
Qiazol lysis reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
QIAzol Lysis Reagent is a mono-phasic solution of phenol and guanidine isothiocyanate that is used for the isolation of total RNA from a variety of biological samples, including cells, tissues, and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy mini kit, by Qiagen (2 mentions)
Miscript 2 rt kit, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2 system, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Applied biosystems 7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mini vortexer, by Thermo Fisher Scientific (1 mentions)
Mirna specific primer, by Qiagen (1 mentions)
Mirnaeasy mini kit, by Qiagen (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Rt2 mirna first strand kit, by Qiagen (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Qiacube robotic workstation, by Qiagen (1 mentions)
Mirneasy serum plasma kit, by Qiagen (1 mentions)
10 protocols using qiazol lysis reagent
1
Rapid DNA and RNA Extraction from Plant Leaves
2
RNA Extraction from Bladder Tissue
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Bladder tissue biopsies were homogenized as previously described 3 (link), using a Tissue Lyser, (Qiagen, Hilden, Germany) before RNA extraction. Total RNA was extracted using the Qiazol Lysis Reagent® (Thermo Fisher Scientific, Waltham, MA), following the manufacturer’s instructions. The quantity of RNA was determined using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies inc, Wilmington, DE). RNA integrity (RIN) was determined using an Agilent 2100 Bioanalyser (RNA 6000 Nano LabChip kit, Agilent Technologies).
3
Serum miRNA Expression Analysis
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Total RNA was isolated from the serum of patients or cells using the QIAzol™ reagent (Qiagen, Inc.) as previously described (18 (link),19 (link)). The miRNA expression analysis was performed using qPCR analysis as previously reported (18 (link),19 (link)). Briefly, serum samples were mixed at a ratio of 1:10 with QIAzol™lysis reagent and vortexed for 1 min using a mini vortexer (Thermo Fisher Scientific, Inc). Cell pellets (~106 cells) were mixed with 1 ml QIAzol™ reagent (Qiagen, Inc.). The lysates were extracted using CHCl3 and the aqueous phase was further processed, removing phenol and other contaminants, to obtain total RNA enriched in miRNA using the miRNAeasy Mini Kit (Qiagen, Inc.). miRNA levels were determined following conversion of RNA to cDNA using the RT2 miRNA first strand kit (Qiagen, Inc.) followed by amplification of cDNA in the Applied Biosystems 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) using SYBR qPCR reaction mixture and miRNA specific primers (Qiagen, Inc.). Amplification of cDNA was performed under the following conditions: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec (18 (link),19 (link)). U6 spliceosomal RNA served as an internal control, and data was quantified using the comparative Cq method (20 (link)).
4
Quantitative Analysis of miRNA Expression
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Cells were lysed with QIAzol Lysis Reagent (Life Technologies) and stored at -20 °C. Total RNA was extracted from hiPSCs-derived TSC and control neuron lysates using the miRNeasy mini kit (reference 217,004, Qiagen, Germany). For each sample, 1 µg of total RNA was reverse transcribed using the miScript II RT Kit (Qiagen). For qPCR analysis, QuantiTect SYBR Green PCR kit (Qiagen, Germany) was employed and samples were run on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) following the manufacturer’s instructions. All reactions were performed in triplicate for each sample. The relative expression levels of the miRNAs and other genes were calculated using the 2−ΔΔCT method [27 (link)], and the data were normalised to GAPDH and Colrf43. The primer sequences for all genes examined in the current study are listed in Additional file 1 : Table S1.
5
Robust miRNA Purification and Quantification
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Total RNA including miRNA from serum and brain tissue samples was purified by using miRNeasy Serum/Plasma Kit (Qiagen, Germany) and miRNeasy Mini Kit (Qiagen, Germany), respectively. All purification procedures were performed according to attached protocols combined with the use of a QIAcube robotic workstation (Qiagen, Germany) for automated purification of total RNA including miRNA. Synthetic ath-miR-159a miRNA mimic (Life Technologies, CA, USA) was spiked into QIAzol Lysis Reagent to a final concentration of 4 fmol/ml in order to control quality of the RNA purification, complementary DNA (cDNA) synthesis, and PCR amplification in the qPCR experiment. Purity and concentration of purified RNA samples were measured with the use of NanoDrop spectrophotometer (Thermo Scientific, MA, USA). Samples with good quality were taken for further RT-qPCR analysis.
6
RNA Extraction and qRT-PCR Analysis
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H9c2, NMCMs, and hiPSC-CMs were collected into QIAzol Lysis Reagent or Trizol Reagent (Invitrogen) and total RNA was extracted using Direct-zol RNA Miniprep and Microprep (Zymo Research, CA, USA), respectively. Heart samples were first homogenized in QIAzol Lysis Reagent using the Qiagen TissueLyser LT Bead Mill (QIAGEN, Venlo, Netherlands) and total RNA was thereafter extracted using Direct-zol RNA Miniprep. RNA was reverse-transcribed to cDNA (PrimeScript RT reagent kit, Takara Clontech) and quantitative real-time polymerase chain reaction (RT-PCR) was performed with SYBR Green protocols (Kapa Biosystems, MA, USA) and a real-time PCR detection system (Applied Biosystems 7300 Real-Time PCR system) (Rinne et al, 2017 (link); Kadiri et al, 2021 (link)). Target gene expression was normalized to a housekeeping gene (ribosomal protein S18; RPS18, glyceraldehyde-3-phosphate dehydrogenase; GAPDH or β-actin; ACTB) using the comparative ΔCt method and results are presented as relative transcript levels (2-ΔΔCt). Primer sequences are presented in Appendix Tables S4 –S6 .
7
Comprehensive RNA Extraction and Detection Protocol
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TRIzol Reagent (Invitrogen, USA) can be used for total RNA extraction from tissues and cells. Total RNA in serum and exosomes was extracted with QIAzol Lysis Reagent (Invitrogen, USA). The miScript II RT Kit (Qiagen, Germany) can be used to reverse transcribe mRNAs, and the miScript SYBR Green PCR Kit (Qiagen, Germany) can be used to detect them. Similarity, the miScript II RT Kit (Qiagen, Germany) is used for miRNA reverse transcription, and the SYBR Green PCR Kit (Qiagen, Germany) can be used to detect reverse transcriptional mRNA. Quantitative analyses can use CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were repeated three times. The internal reference of mRNAs is GAPDH. The internal parameter of miRNAs is U6. Sangon (Shanghai, China) (Supplementary Table S2 ) designed and synthesized primers for mRNAs. The primers for miRNA detection were purchased from Qiagen.
8
Extracting and Amplifying RNA from Caco-2/15 Cells
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Total RNA was extracted from differentiated CTL and CFTR-/- Caco-2/15 cells using QIAzol lysis reagent (Invitrogen, Thermo Fisher scientific, Burlington, ON, Canada) and reverse transcribed to generate cDNA. This was amplified by PCR using Taq polymerase (Feldan Bio, Quebec, QC, Canada) according to the manufacturer’s instructions. GAPDH (as internal control) and CFTR as described previously [17 (link)].
9
Transcriptional Profiling of TOS1-CSCs
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Total RNA of TOS1-CSC and TOS1 cell lines was extracted by using Qiazol Lysis Reagent (Invitrogen, USA). Reverse transcription and quantitative Real Time PCR (RT-qPCR) analyses were carried out as described using specific primers and probes (Table 2 ) which were designed by IDT integrated DNA technologies, following the manufacturer’s protocol. GAPDH was used as internal control. RT-qPCR was conducted using TaqMan Real-Time PCR Master Mix (Resnova, Roma, Italy) on a Rotor-Gene Q real-time PCR cycler (QIAGEN, Hilden, Germany). All points for standard curves and unknown samples were performed in triplicate. Student‘s t-test was used to determine the differences between TOS1-CSCs and TOS1. A p-value of <0.001 was considered statistically significant.
10
Dorsal Hippocampus RNA Extraction
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Dorsal hippocampus was quickly dissected, placed into RNA later (Qiagen Valencia, CA), or frozen in liquid nitrogen. Tissues were homogenized in QIAzol Lysis Reagent (Invitrogen) and mixed in chloroform. RNA in aqueous-phase was separated in phase-lock tubes following centrifugation at 14,000g for 15 min. RNA was extracted using RNAeasy kit (Qiagen) according to the manufacturer’s instruction. Samples were DNase treated using the RNase-Free DNase kit (Qiagen) off-column. Samples were ethanol precipitated and resuspended in RNAse-free water. Samples with an OD 260/280 and OD 260/230 ratio close to 2.0 and RNA integrity number (RIN) above 8 were selected for library preparation.
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